Masako Nomaguchi, Doi Naoya, Fujiwara Sachi and Akio Adachi : Macaque-tropic HIV-1 derivatives: a novel experimental approach to understand viral replication and evolution in vivo., 2011.
Academic Paper (Judged Full Paper):
1.
Takaaki Koma, Naoya Doi, Bao Quoc Le, Tomoyuki Kondo, Mitsuki Ishizue, Chiaki Tokaji, Chizuko Tsukada, Akio Adachi and Masako Nomaguchi : Involvement of a Rarely Used Splicing SD2b Site in the Regulation of HIV-1 mRNA Production as Revealed by a Growth-Adaptive Mutation., Viruses, Vol.15, No.12, 2424, 2023.
(Summary)
We have previously reported an HIV-1 mutant designated NL-Y226tac that expresses Vif at an ultra-low level, being replication-defective in high-APOBEC3G cells, such as H9. It carries a synonymous mutation within the splicing SA1 site relative to its parental clone. In order to determine whether a certain mutant(s) emerges during multi-infection cycles, we maintained H9 cells infected with a relatively low or high input of NL-Y226tac for extended time periods. Unexpectedly, we reproducibly identified a g5061a mutation in the SD2b site in the two independent long-term culture experiments that partially increases Vif expression and replication ability. Importantly, the adaptive mutation g5061a was demonstrated to enhance mRNA production by activation of the SA1 site mediated through increasing usage of a rarely used SD2b site. In the long-term culture initiated by a high virus input, we additionally found a Y226Fttc mutation at the original Y226tac site in SA1 that fully restores Vif expression and replication ability. As expected, the adaptive mutation Y226Fttc enhances mRNA production through increasing the splicing site usage of SA1. Our results here revealed the importance of the SD2b nucleotide sequence in producing mRNA involved in the HIV-1 adaptation and of mutual antagonism between Vif and APOBEC3 proteins in HIV-1 adaptation/evolution and survival.
Takaaki Koma, Naoya Doi, Akihiro Suzuki, Kentaro Nagamatsu, Takeshi Yasui, Koji Yasutomo, Akio Adachi, Takeo Minamikawa and Masako Nomaguchi : Major target for UV-induced complete loss of HIV-1 infectivity: A model study of single-stranded RNA enveloped viruses, Frontiers in Virology, Vol.2, 994842, 2022.
Takaaki Koma, Naoya Doi, Mai Takemoto, Kyosuke Watanabe, Hideki Yamamoto, Satoshi Nakashima, Akio Adachi and Masako Nomaguchi : The Expression Level of HIV-1 Vif Is Optimized by Nucleotide Changes in the Genomic SA1D2prox Region during the Viral Adaptation Process., Viruses, Vol.13, No.10, 2021.
(Summary)
HIV-1 Vif plays an essential role in viral replication by antagonizing anti-viral cellular restriction factors, a family of APOBEC3 proteins. We have previously shown that naturally-occurring single-nucleotide mutations in the SA1D2prox region, which surrounds the splicing acceptor 1 and splicing donor 2 sites of the HIV-1 genome, dramatically alter the Vif expression level, resulting in variants with low or excessive Vif expression. In this study, we investigated how these HIV-1 variants with poor replication ability adapt and evolve under the pressure of APOBEC3 proteins. Adapted clones obtained through adaptation experiments exhibited an altered replication ability and Vif expression level compared to each parental clone. While various mutations were present throughout the viral genome, all replication-competent adapted clones with altered Vif expression levels were found to bear them within SA1D2prox, without exception. Indeed, the mutations identified within SA1D2prox were responsible for changes in the Vif expression levels and altered the splicing pattern. Moreover, for samples collected from HIV-1-infected patients, we showed that the nucleotide sequences of SA1D2prox can be chronologically changed and concomitantly affect the Vif expression levels. Taken together, these results demonstrated the importance of the SA1D2prox nucleotide sequence for modulating the Vif expression level during HIV-1 replication and adaptation.
Takaaki Koma, Masaru Yokoyama, Osamu Kotani, Naoya Doi, Nina Nakanishi, Hayato Okubo, Shun Adachi, Akio Adachi, Hironori Sato and Masako Nomaguchi : Species-specific valid ternary interactions of HIV-1 Env-gp120, CD4, and CCR5 as revealed by an adaptive single-amino acid substitution at the V3 loop tip., Journal of Virology, 2021.
(Summary)
Understanding molecular bases for viral entry into cells leads to the elucidation of one of major viral survival strategies, and thus to the development of new effective antiviral measures. As experimentally shown recently, HIV-1 is highly mutable and adaptable in growth-restrictive cells such as those of macaque origin. HIV-1 initiates its infection by sequential interactions of Env-gp120 with two cell surface receptors, CD4 and CCR5. A recent epoch-making structural study has disclosed that CD4-induced conformation of gp120 is stabilized upon binding of CCR5 to the CD4-gp120 complex, whereas its biological significance remains totally unknown. Here, from a series of mutations found in our extensive studies, we identified a single-amino acid adaptive mutation at the V3 loop tip of Env-gp120 critical for its interaction with both CD4 and CCR5 in a host cell species-specific way. This remarkable finding would certainly provoke and accelerate studies to precisely clarify the HIV-1 entry mechanism.
Naoya Doi, Takaaki Koma, Akio Adachi and Masako Nomaguchi : Expression Level of HIV-1 Vif Can Be Fluctuated by Natural Nucleotide Variations in the vif-Coding and Regulatory SA1D2prox Sequences of the Proviral Genome., Frontiers in Microbiology, Vol.10, 2019.
Naoya Doi, Takaaki Koma, Akio Adachi and Masako Nomaguchi : Role for Gag-CA Interdomain Linker in Primate Lentiviral Replication., Frontiers in Microbiology, Vol.10, 2019.
(Summary)
-modulator to optimize the Gag-related viral replication process. We also have noted, during the course of conducting the research project, that HIV-1 and SIVmac, belonging to distinct primate lentiviral lineages, share a similarly biologically active linker region with each other. In this brief article, we summarize and report the results obtained by mutational studies that are relevant to the functional significance of the interdomain linker of HIV/SIV Gag-CA. Based on this investigation, we discuss about the future directions of the research in this line.
Takaaki Koma, Osamu Kotani, Kei Miyakawa, Akihide Ryo, Masaru Yokoyama, Naoya Doi, Akio Adachi, Hironori Sato and Masako Nomaguchi : Allosteric regulation of HIV-1 capsid structure for Gag assembly, virion production, and viral infectivity by a disordered interdomain linker., Journal of Virology, 2019.
(Summary)
HIV-1 particle production and infection are highly ordered processes. Viral Gag proteins play a central role in the assembly and disassembly of viral molecules. Of these, capsid protein (CA) is a major contributor to the Gag-Gag interactions. CA consists of two structured domains, i.e., N-terminal (NTD) and C-terminal (CTD) domains, connected by an unstructured domain named interdomain linker. While multiple regions in the NTD and CTD domains are reported to play roles in virion morphogenesis and infectivity, the roles of the linker region in Gag assembly and virus particle formation remain elusive. In this report, we show by biological and molecular analyses that the linker region functions as an intramolecular modulator to tune Gag assembly, virion production, and viral infectivity. Our study thus illustrates a hitherto unrecognized mechanism, an allosteric regulation of CA structure by the disordered protein element, for HIV-1 replication.
Naoya Doi, Masaru Yokoyama, Takaaki Koma, Osamu Kotani, Hironori Sato, Akio Adachi and Masako Nomaguchi : Concomitant Enhancement of HIV-1 Replication Potential and Neutralization-Resistance in Concert With Three Adaptive Mutations in Env V1/C2/C4 Domains., Frontiers in Microbiology, Vol.10, 2019.
(Summary)
adapts its Env to macaque cells with strongly replication-restrictive nature for HIV-1. While a single and two mutations gave a significantly enhanced replication phenotype in a macaque cell line and also in human cell lines that stably express either human CD4 or macaque CD4, the virus simultaneously carrying the three adaptive mutations always grew best. Entry kinetics of parental and triple mutant viruses were similar, whereas the mutant was significantly more readily inhibited for its infectivity by soluble CD4 than parental virus. Furthermore, molecular dynamics simulations of the Env ectodomain (gp120 and gp41 ectodomain) bound with CD4 suggest that the three mutations increase binding affinity of Env for CD4 in solution. Thus, it is quite likely that the affinity for CD4 of the mutant Env is enhanced relative to the parental Env. Neutralization sensitivity of the triple mutant to CD4 binding site antibodies was not significantly different from that of parental virus, whereas the mutant exhibited a considerably higher resistance against neutralization by a CD4-induced epitope antibody and Env trimer-targeting V1/V2 antibodies. These results suggest that the three adaptive mutations cooperatively promote viral growth via increased CD4 affinity, and also that they enhance viral resistance to several neutralization antibodies by changing the Env-trimer conformation. In total, we have verified here an HIV-1 adaptation pathway in host cells and individuals involving Env derived from a lab-adapted and highly neutralization-sensitive clone.
Naoya Doi, Tomoyuki Miura, Hiromi Mori, Hiromi Sakawaki, Takaaki Koma, Akio Adachi and Masako Nomaguchi : CXCR4- and CCR5-Tropic HIV-1 Clones Are Both Tractable to Grow in Rhesus Macaques., Frontiers in Microbiology, Vol.9, 2018.
(Summary)
genetic manipulations and viral cell-adaptations, we have successfully generated a series of HIV-1 derivatives with CXCR4-tropism or CCR5-tropism that grow in macaque cells to various degrees. Of these viruses, those with best replicative potentials can grow comparably with a pathogenic SIVmac in macaque cells by counteracting major restriction factors TRIM5, APOBEC3, and tetherin proteins. In this study, rhesus macaques were challenged with CXCR4-tropic (MN4/LSDQgtu) or CCR5-tropic (gtu + A4CI1) virus. The two viruses were found to productively infect rhesus macaques, being rhesus macaque-tropic HIV-1 (HIV-1rmt). However, plasma viral RNA was reduced to be an undetectable level in infected macaques at 5-6 weeks post-infection and thereafter. While replicated similarly well in rhesus peripheral blood mononuclear cells, MN4/LSDQgtu grew much better than gtu + A4CI1 in the animals. To the best of our knowledge, this is the first report demonstrating that HIV-1 derivatives (variants) grow in rhesus macaques. These viruses certainly constitute firm bases for generating HIV-1rmt clones pathogenic for rhesus monkeys, albeit they grow more poorly than pathogenic SIVmac and SHIV clones reported to date.
Shoko Nakanishi, Sakimi Watanabe, Naoya Doi, Takaaki Koma, Akio Adachi and Masako Nomaguchi : Virological characterization of HIV-1 CA-NTD mutants constructed in a virus-lineage reflected manner., The Journal of Medical Investigation : JMI, Vol.65, No.1.2, 110-115, 2018.
(Summary)
Capsid (CA) protein is a major virion-constituent of all retroviruses including human immunodeficiency virus type 1 (HIV-1), and is essential for early and late phases in viral replication cycle through interaction with numerous cellular factors. In particular, N-terminal domain (NTD) of HIV-1 CA has been frequently and well reported to bind to various host cell proteins that considerably affect viral replication potential. In this study, in order to better define biological bases of the CA-NTD for HIV-1 replication, we performed an extensive mutational analysis in an unprecedented manner. By aligning CA-NTD sequences derived from representative infectious molecular clones of HIV-1, HIV-2, and simian immunodeficiency virus isolated from the rhesus macaque (SIVmac), a number of amino acids specific to HIV-1 were selected, and were replaced with those from SIVmac at the corresponding sites. Mutant viruses thus generated were then examined for multi-cycle infectivity, single-cycle infectivity, and ability to produce progeny virions. While some CA-NTD mutations affected viral replication ability to varying degrees, those in helix 7 abolished viral growth potential without exception. These results highlight functional importance of non-conserved amino acids in helix 7, and give new insights into functionality of HIV-1 CA-NTD. J. Med. Invest. 65:110-115, February, 2018.
Masako Nomaguchi, Naoya Doi, Takaaki Koma and Akio Adachi : Complete Genome Sequences of Human Immunodeficiency Type 1 Viruses Genetically Engineered To Be Tropic for Rhesus Macaques., Genome Announcements, Vol.5, No.39, 2017.
(Summary)
We have constructed two human immunodeficiency type 1 (HIV-1) derivatives, CXCR4 tropic and CCR5 tropic, that replicate in rhesus macaques. They are genetically engineered to be resistant to macaque restriction factors against HIV-1, including TRIM5, APOBEC3, and tetherin proteins. The two HIV-1 variants described here are fundamental clones aiming for rhesus infection studies of HIV-1.
Naoya Doi, Yosuke Sakai, Akio Adachi and Masako Nomaguchi : Generation and characterization of new CCR5-tropic HIV-1rmt clones, The Journal of Medical Investigation : JMI, Vol.64, No.3,4, 272-279, 2017.
(Summary)
To develop effective non-human primate models for coping with numerous HIV-1/AIDS studies, rhesus macaque-tropic HIV-1 (HIV-1rmt) clones with a variety of biological properties are required. Such clones, if available, are powerful tools to experimentally elucidate HIV-1 replication and pathogenicity in host individuals, and also to develop anti-HIV-1 drugs/vaccines. However, only limited numbers of HIV-1rmt clones have been currently reported. In the present study, we generated new HIV-1rmt clones carrying various CCR5-tropic env (envelope) genes by standard recombinant DNA and intracellular homologous recombination techniques. Resultant virus clones contain the env sequences derived from an AIDS-inducible laboratory or two clinically isolated viral strains. We further constructed their variant clones bearing N160K, S304G, or G310R mutation in Env that potentially can change the viruses to better grow. Newly generated clones were analyzed for their virological properties such as Env expression, single-cycle infectivity, and multi-cycle replication ability. Out of a number of new clones examined, two were found to grow better in macaque cells than the previously constructed clone used for comparison. Our study described here constitutes the initial and essential step towards obtaining CCR5-tropic HIV-1rmt clones useful for various basic and clinical research projects on infected individuals. J. Med. Invest. 64: 272-279, August, 2017.
Yasuyuki Miyazaki, Naoya Doi, Takaaki Koma, Akio Adachi and Masako Nomaguchi : Novel In Vitro Screening System Based on Differential Scanning Fluorimetry to Search for Small Molecules against the Disassembly or Assembly of HIV-1 Capsid Protein., Frontiers in Microbiology, Vol.8, 1413, 2017.
(Summary)
Varieties of in vitro systems have been used to study biochemical properties of human immunodeficiency virus Gag-capsid protein (HIV Gag-CA). Recently, we have comparatively characterized HIV-1 and HIV-2 Gag-CA proteins using such technology, and have demonstrated that the NaCl-initiated CA-polymerization in vitro and the stability of CA N-terminal domain as judged by differential scanning fluorimetry (DSF) are inversely correlated. In this study, we found that ZnCl2 works as a competent initiator of the in vitro HIV-1 CA-polymerization at much lower concentrations than those of NaCl frequently used for the polymerization initiation. We also showed by DSF assays that ZnCl2 highly destabilize HIV-1 CA. Furthermore, PF74, a well-known inducer of premature HIV-1 uncoating in infected cells, was demonstrated to unusually promote the HIV-1 CA-disassembly in the presence of ZnCl2 as revealed by DSF assays. Taken together, we conclude that the DSF method may be useful as an efficient monitoring system to screen anti-HIV-1 CA molecules.
Yasuyuki Miyazaki, Ariko Miyake, Noya Doi, Takaaki Koma, Tsuneo Uchiyama, Akio Adachi and Masako Nomaguchi : Comparison of Biochemical Properties of HIV-1 and HIV-2 Capsid Proteins., Frontiers in Microbiology, Vol.8, No.1, 1082, 2017.
(Summary)
Timely disassembly of viral core composed of self-assembled capsid (CA) in infected host cells is crucial for retroviral replication. Extensive in vitro studies to date on the self-assembly/disassembly mechanism of human immunodeficiency virus type 1 (HIV-1) CA have revealed its core structure and amino acid residues essential for CA-CA intermolecular interaction. However, little is known about in vitro properties of HIV-2 CA. In this study, we comparatively analyzed the polymerization properties of bacterially expressed HIV-1 and HIV-2 CA proteins. Interestingly, a much higher concentration of NaCl was required for HIV-2 CA to self-assemble than that for HIV-1 CA, but once the polymerization started, the reaction proceeded more rapidly than that observed for HIV-1 CA. Analysis of a chimeric protein revealed that N-terminal domain (NTD) is responsible for this unique property of HIV-2 CA. To further study the molecular basis for different in vitro properties of HIV-1 and HIV-2 CA proteins, we determined thermal stabilities of HIV-1 and HIV-2 CA NTD proteins at several NaCl concentrations by fluorescent-based thermal shift assays. Experimental data obtained showed that HIV-2 CA NTD was structurally more stable than HIV-1 CA NTD. Taken together, our results imply that distinct in vitro polymerization abilities of the two CA proteins are related to their structural instability/stability, which is one of the decisive factors for viral replication potential. In addition, our assay system described here may be potentially useful for searching for anti-CA antivirals against HIV-1 and HIV-2.
Yosuke Sakai, Naoya Doi, Yasuyuki Miyazaki, Akio Adachi and Masako Nomaguchi : Phylogenetic Insights into the Functional Relationship between Primate Lentiviral Reverse Transcriptase and Accessory Proteins Vpx/Vpr., Frontiers in Microbiology, Vol.7, 2016.
(Summary)
The efficiency of reverse transcription to synthesize viral DNA in infected cells greatly influences replication kinetics of retroviruses. However, viral replication in non-dividing cells such as resting T cells and terminally differentiated macrophages is potently and kinetically restricted by a host antiviral factor designated SAMHD1 (sterile alpha motif and HD-domain containing protein 1). SAMHD1 reduces cellular deoxynucleoside triphosphate (dNTP) pools and affects viral reverse transcription step. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) have Vpx or Vpr to efficiently degrade SAMHD1. Interestingly, the reverse transcriptase (RT) derived from HIV-1 that encodes no anti-SAMHD1 proteins has been previously demonstrated to uniquely exhibit a high enzymatic activity. It is thus not irrational to assume that some viruses may have acquired or lost the specific RT property to better adapt themselves to the low dNTP environments confronted in non-dividing cells. This adaptation process may probably be correlated with the SAMHD1-antagonizing ability by viruses. In this report, we asked whether such adaptive events can be inferable from Vpx/Vpr and RT phylogenetic trees overlaid with SAMHD1-degrading capacity of Vpx/Vpr and with kinetic characteristics of RT. Resultant two trees showed substantially similar clustering patterns, and therefore suggested that the properties of RT and Vpx/Vpr can be linked. In other words, HIV/SIVs may possess their own RT proteins to adequately react to various dNTP circumstances in target cells.
Yosuke Sakai, Ariko Miyake, Naoya Doi, Hikari Sasada, Yasuyuki Miyazaki, Akio Adachi and Masako Nomaguchi : Expression Profiles of Vpx/Vpr Proteins Are Co-related with the Primate Lentiviral Lineage., Frontiers in Microbiology, Vol.7, 2016.
(Summary)
Viruses of human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency virus (SIV) lineages carry a unique accessory protein called Vpx. Vpx is essential or critical for viral replication in natural target cells such as macrophages and T lymphocytes. We have previously shown that a poly-proline motif (PPM) located at the C-terminal region of Vpx is required for its efficient expression in two strains of HIV-2 and SIVmac, and that the Vpx expression levels of the two clones are significantly different. Notably, the PPM sequence is conserved and confined to Vpx and Vpr proteins derived from certain lineages of HIV-2/SIVs. In this study, Vpx/Vpr proteins from diverse primate lentiviral lineages were experimentally and phylogenetically analyzed to obtain the general expression picture in cells. While both the level and PPM-dependency of Vpx/Vpr expression in transfected cells varied among viral strains, each viral group, based on Vpx/Vpr amino acid sequences, was found to exhibit a characteristic expression profile. Moreover, phylogenetic tree analyses on Gag and Vpx/Vpr proteins gave essentially the same results. Taken together, our study described here suggests that each primate lentiviral lineage may have developed a unique expression pattern of Vpx/Vpr proteins for adaptation to its hostile cellular and species environments in the process of viral evolution.
Masako Nomaguchi, N. Doi, Y. Sakai, H. Ode, Y. Iwatani, T. Ueno, Y. Matsumoto, Y. Miyazaki, T. Masuda and Akio Adachi : Natural single-nucleotide variations in the HIV-1 genomic SA1prox region can alter viral replication ability by regulating Vif expression levels., Journal of Virology, Vol.90, No.9, 4563-4578, 2016.
(Summary)
We previously found that natural single-nucleotide variations located within a proximal region of splicing acceptor 1 (SA1prox) in the HIV-1 genome could alter the viral replication potential and mRNA expression pattern, especially the vif mRNA level. Here, we studied the virological and molecular basis of nucleotide sequence variations in SA1prox for alterations of viral replication ability. Consistent with our previous findings, variant clones indeed expressed Vif at different levels and grew distinctively in cells with various APOBEC3G expression levels. Similar effects were observed for natural variations found in HIV-2 SA1prox, suggesting the importance of the SA1prox sequence. To define nucleotides critical for the regulation of HIV-1 Vif expression, effects of natural SA1prox variations newly found in the HIV Sequence Compendium database on vif mRNA/Vif protein levels were examined. Seven out of nine variations were found to produce Vif at lower, higher, or more excessive levels than wild-type NL4-3. Combination experiments of variations giving distinct Vif levels suggested that the variations mutually affected vif transcript production. While low and high producers of Vif grew in an APOBEC3G-dependent manner, excessive expressers always showed an impeded growth phenotype due to defects in single-cycle infectivity and/or virion production levels. The phenotype of excessive expressers was not due primarily to inadequate expression of Tat or Rev, although SA1prox variations altered the overall HIV-1 mRNA expression pattern. Collectively, our results demonstrate that HIV SA1prox regulates Vif expression levels and suggest a relationship between SA1prox and viral adaptation/evolution given that variations occurred naturally. While human cells possess restriction factors to inhibit HIV-1 replication, HIV-1 encodes antagonists to overcome these barriers. Conflicts between host restriction factors and viral counterparts are critical driving forces behind mutual evolution. The interplay of cellular APOBEC3G and viral Vif proteins is a typical example. Here, we demonstrate that naturally occurring single-nucleotide variations in the proximal region of splicing acceptor 1 (SA1prox) of the HIV-1 genome frequently alter Vif expression levels, thereby modulating viral replication potential in cells with various ABOBEC3G levels. The results of the present study reveal a previously unidentified and important way for HIV-1 to compete with APOBEC3G restriction by regulating its Vif expression levels. We propose that SA1prox plays a regulatory role in Vif counteraction against APOBEC3G in order to contribute to HIV-1 replication and evolution, and this may be applicable to other primate lentiviruses.
(Keyword)
Alternative Splicing / Amino Acid Sequence / Base Sequence / Cell Line / Codon / Gene Expression Regulation, Viral / Gene Order / Genome, Viral / HIV-1 / Humans / Nucleic Acid Conformation / Polymorphism, Single Nucleotide / RNA Splice Sites / RNA, Viral / Recombination, Genetic / Transcription, Genetic / Virus Replication / vif Gene Products, Human Immunodeficiency Virus
M. Yokoyama, Masako Nomaguchi, N. Doi, T. Kanda, Akio Adachi and H. Sato : In silico analysis of HIV-1 Env-gp20 reveals structural bases for viral adaptation in growth-restrictive cells., Frontiers in Microbiology, Vol.7, No.110, 2016.
(Summary)
Variable V1/V2 and V3 loops on human immunodeficiency virus type 1 (HIV-1) envelope-gp120 core play key roles in modulating viral competence to recognize two infection receptors, CD4 and chemokine-receptors. However, molecular bases for the modulation largely remain unclear. To address these issues, we constructed structural models for a full-length gp120 in CD4-free and -bound states. The models showed topologies of gp120 surface loop that agree with those in reported structural data. Molecular dynamics simulation showed that in the unliganded state, V1/V2 loop settled into a thermodynamically stable arrangement near V3 loop for conformational masking of V3 tip, a potent neutralization epitope. In the CD4-bound state, however, V1/V2 loop was rearranged near the bound CD4 to support CD4 binding. In parallel, cell-based adaptation in the absence of anti-viral antibody pressures led to the identification of amino acid substitutions that individually enhance viral entry and growth efficiencies in association with reduced sensitivity to CCR5 antagonist TAK-779. Notably, all these substitutions were positioned on the receptors binding surfaces in V1/V2 or V3 loop. In silico structural studies predicted some physical changes of gp120 by substitutions with alterations in viral replication phenotypes. These data suggest that V1/V2 loop is critical for creating a gp120 structure that masks co-receptor binding site compatible with maintenance of viral infectivity, and for tuning a functional balance of gp120 between immune escape ability and infectivity to optimize HIV-1 replication fitness.
Tahmina Sultana, E Emi Nakayama, Satoshi Tobita, Masaru Yokoyama, Yohei Seki, Akatsuki Saito, Masako Nomaguchi, Akio Adachi, Hirofumi Akari, Hironori Sato and Tatsuo Shioda : Novel mutant human immunodeficiency virus type 1 strains with high degree of resistance to cynomolgus macaque TRIMCyp generated by random mutagenesis., The Journal of General Virology, Vol.97, No.4, 963-976, 2016.
(Summary)
Old World monkey TRIM5 strongly suppresses human immunodeficiency virus type 1 (HIV-1) replication. A fusion protein comprising cynomolgus macaque (CM) TRIM5 and cyclophlin A (CM TRIMCyp) also potently suppresses HIV-1 replication. However, CM TRIMCyp fails to suppress a mutant HIV-1 that encodes a mutant capsid protein containing a SIVmac239-derived loop between helices 4 and 5 (L4/5). There are seven amino acid differences between L4/5 of HIV-1 and SIVmac239. Here, we investigated the minimum numbers of amino acid substitutions that would allow HIV-1 to evade CM TRIMCyp-mediated suppression. We performed random PCR mutagenesis to construct a library of HIV-1 variants containing mutations in L4/5, and then we recovered replication-competent viruses from CD4+ MT4 cells that expressed high levels of CM TRIMCyp. CM TRIMCyp-resistant viruses were obtained after three rounds of selection in MT4 cells expressing CM TRIMCyp and these were found to contain four amino acid substitutions (H87R, A88G, P90D, and P93A) in L4/5. We then confirmed that these substitutions were sufficient to confer CM TRIMCyp resistance to HIV-1. In a separate experiment using a similar method, we obtained novel CM TRIM5-resistant HIV-1 strains after six rounds of selection and rescue. Analysis of these mutants revealed that V86A and G116E mutations in the capsid region conferred partial resistance to CM TRIM5 without substantial fitness cost propagated in MT4 cells expressing CM TRIM5. These results confirmed and further extended the previous notion that CM TRIMCyp and CM TRIM5 recognize the HIV-1 capsid in different manners.
Naoya Doi, Yosuke Sakai, Yasuyuki Miyazaki, Akio Adachi and Masako Nomaguchi : Single-amino acid mutation 66SR in Gag-matrix enhances viral single-cycle infectivity of R5-tropic HIV-1rmt., The Journal of Medical Investigation : JMI, Vol.62, No.3-4, 228-232, 2015.
(Summary)
We recently constructed two rhesus macaque-tropic human immunodeficiency virus type 1 (HIV-1rmt) clones with CXCR4 or CCR5 tropism, but a CCR5-tropic HIV-1rmt clone grew more poorly than a CXCR4-topic clone. It has been demonstrated that interaction between viral Gag-matrix (MA) and Env-gp41 cytoplasmic tail is important for virion-incorporation of Env. Concordantly, Gag-MA mutations (62QR and 66SR) that rescue defects in virion-incorporation of Env/viral replication were reported. In this study, we analyzed effects of these Gag-MA mutations on R5-tropic HIV-1rmt replication potentials. While introduction of 62QR into three HIV-1rmt clones tested reduced their multi-cycle replication ability in rhesus lymphocytes or abolish single-cycle infectivity for luciferase reporter cells, three R5-tropic HIV-1rmt clones carrying 66SR exhibited similar growth kinetics to those of their parental clones. One such clone, 66SR+5gtu, appeared to induce stronger cytopathic effects than parental clone 5gtu. We therefore investigated effects of 66SR mutation on viral replication in more detail. Single-cycle infectivity of 66SR+5gtu was enhanced relative to that of 5gtu, but 66SR+5gtu virion production was significantly decreased compared to the 5gtu level. Gag-MA 66SR mutation may be useful to improve growth potentials of the R5-tropic HIV-1rmt clones.
Masako Nomaguchi, Emi E. Nakayama, Masaru Yokoyama, Naoya Doi, Tatsuhiko Igarashi, Tatsuo Shioda, Hironori Sato and Akio Adachi : Distinct combinations of amino acid substitutions in N-terminal domain of Gag-capsid afford HIV-1 resistance to rhesus TRIM5α., Microbes and Infection, Vol.16, No.11, 936-944, 2014.
(Summary)
TRIM5α is a potent anti-retroviral factor that interacts with viral capsid (CA) in a species-specific manner. Recently, we and others reported generation of two distinct HIV-1 CAs that effectively overcome rhesus TRIM5α-imposed species barrier. In this study, to directly compare the effect of different mutations in the two HIV-1 CAs on evasion from macaque TRIM5-restriction, we newly generated macaque-tropic HIV-1 (HIV-1mt) proviral clones carrying the distinct CAs in the same genomic backbone, and examined their replication abilities in macaque TRIM5-overexpressing human cells and in rhesus cells. Comparative analysis of amino acid sequences and homology modeling-based structures revealed that, while both CAs gained some mutated amino acids with similar physicochemical properties, their overall appearances of N-terminal domains were different. Experimentally, the two CAs exhibited incomplete TRIM5α-resistance relative to SIVmac239 CA and different degrees of susceptibility to various TRIM5 proteins. Finally, two HIV-1mt clones carrying a different combination of the CA mutations were found to grow to a comparable extent in established and primary rhesus cells. Our data show that there could be some distinct CA patterns to confer significant TRIM5-resistance on HIV-1.
Masako Nomaguchi, Naoya Doi and Akio Adachi : Virological characterization of HIV-2 vpx gene mutants in various cell systems., Microbes and Infection, Vol.16, No.8, 695-701, 2014.
(Summary)
Requirement of intrinsically disordered protein Vpx for HIV-2 replication is cell-type dependent. To define Vpx-dependent conditions, replication ability of HIV-2 vpx mutants was analyzed in various cell lines that differ in cellular type, differentiation state and/or expression level of anti-HIV-1 SAMHD1 degraded by Vpx. Induction of Vpx-sensitive anti-HIV-2 state was not always associated with SAMHD1 expression. Compared with our previous data in lymphocytic cells, growth-defectiveness of the vpx mutants in differentiated THP-1 cells, a newly established multi-cycle infection system, was considerably different. Taken together, our results suggest that Vpx plays cell-type dependent role through its undetermined structure and/or function.
Masako Nomaguchi, Ariko Miyake, Naoya Doi, Sachi Fujiwara, Yasuyuki Miyazaki, Yasuko Tsunetsugu-Yokota, Masaru Yokoyama, Hironori Sato, Takao Masuda and Akio Adachi : Natural single-nucleotide polymorphisms in the 3' region of the HIV-1 pol gene modulate viral replication ability., Journal of Virology, Vol.88, No.8, 4145-4160, 2014.
(Summary)
Viruses have the plasticity to adapt themselves under various constraints. HIV-1 can mutate and evolve in growth-restrictive cells by acquiring adaptive changes in its genome. We have previously identified some growth-enhancing mutations in a narrow region of the IN-coding sequence, in which a number of cis-acting elements are located. We now focus on the virological significance of this pol gene region and the mechanistic basis underlying its effects on viral replication. We have found several naturally occurring synonymous mutations within this region that alter viral replication potentials. The effects caused by these natural single-nucleotide polymorphisms are linked to the definite expression patterns of viral mRNAs. We show here that the nucleotide sequence of the pol gene (nucleotides 4895 to 4929 for HIV-1 NL4-3) plays an important role in HIV-1 replication by modulating viral gene expression.
(Keyword)
Base Sequence / HIV Infections / HIV-1 / Humans / Molecular Sequence Data / Polymorphism, Single Nucleotide / Virus Replication / pol Gene Products, Human Immunodeficiency Virus
Naoya Doi, Akio Adachi and Masako Nomaguchi : Growth properties of macaque-tropic HIV-1 clones carrying vpr/vpx genes derived from simian immunodeficiency viruses in place of their vpr regions., The Journal of Medical Investigation : JMI, Vol.61, No.3-4, 374-379, 2014.
(Summary)
We have previously generated a macaque-tropic human immunodeficiency virus type 1 (HIV-1mt) clone designated MN4/LSDQgtu by genetic manipulation from a parental virus that replicates poorly in rhesus macaque cells. In rhesus cell line M1.3S and peripheral blood mononuclear cells (PBMCs), MN4/LSDQgtu grows comparably to a standard simian immunodeficiency virus clone derived from the rhesus macaque (SIVmac239) that can induce the acquired immunodeficiency syndrome (AIDS) in the animals. In this study, we further modified the Vpr-coding region of MN4/LSDQgtu genome by introducing vpr gene of an SIV clone from the greater spot-nosed monkey (SIVgsn166) or vpx gene of SIVmac239 to generate four new clones for determining functional importance of the central genomic area. Furthermore, two clones with an additional Gag-p6 mutation were made to ensure the virion-packaging of Vpx. In addition, accessory gene mutant clones of MN4/LSDQgtu with a frame-shift mutation, including a vpr mutant, were constructed and their growth properties were examined. Infection experiments showed that newly constructed viruses all grew poorly to various degrees in M1.3S cells, relative to MN4/LSDQgtu. Together with the previous data, our results here show that vpr/vpx gene in the appropriate context of HIV-1 genome is critical for viral growth ability.
Ariko Miyake, Mikako Fujita, Haruna Fujino, Ryoko Koga, Sogo Kawamura, Masami Otsuka, Hirotaka Ode, Yasumasa Iwatani, Yosuke Sakai, Naoya Doi, Masako Nomaguchi, Akio Adachi and Yasuyuki Miyazaki : Poly-proline motif in HIV-2 Vpx is critical for its efficient translation., The Journal of General Virology, Vol.95, No.Pt 1, 179-189, 2013.
(Summary)
Human immunodeficiency virus type 2 (HIV-2) carries an accessory protein Vpx that is important for viral replication in natural target cells. In its C-terminal region, there is a highly conserved poly-proline motif (PPM) consisting of seven consecutive prolines, encoded in a poly-pyrimidine tract. We have previously shown that PPM is critical for Vpx expression and viral infectivity. To elucidate the molecular basis underlying this observation, we analysed the expression of Vpx proteins with various PPM mutations by in vivo and in vitro systems. We found that the number and position of consecutive prolines in PPM are important for Vpx expression, and demonstrated that PPM is essential for efficient Vpx translation. Furthermore, mutational analysis to synonymously disrupt the poly-pyrimidine tract suggested that the context of PPM amino acid sequences is required for efficient translation of Vpx. We similarly analysed HIV-1 and HIV-2 Vpr proteins structurally related to HIV-2 Vpx. Expression level of the two Vpr proteins lacking PPM was shown to be much lower relative to that of Vpx, and not meaningfully enhanced by introduction of PPM at the C terminus. Finally, we examined the Vpx of simian immunodeficiency virus from rhesus monkeys (SIVmac), which also has seven consecutive prolines, for PPM-dependent expression. A multi-substitution mutation in the PPM markedly reduced the expression level of SIVmac Vpx. Taken together, it can be concluded that the notable PPM sequence enhances the expression of Vpx proteins from viruses of the HIV-2/SIVmac group at the translational level.
(Keyword)
Amino Acid Motifs / Amino Acid Sequence / Base Sequence / Cell Line / Gene Expression Regulation, Viral / HIV Infections / HIV-2 / Humans / Molecular Sequence Data / Proline / Protein Biosynthesis / vpr Gene Products, Human Immunodeficiency Virus
Masako Nomaguchi, Masaru Yokoyama, Ken Kono, E Emi Nakayama, Tatsuo Shioda, Naoya Doi, Sachi Fujiwara, Akatsuki Saito, Hirofumi Akari, Kei Miyakawa, Akihide Ryo, Hirotaka Ode, Yasumasa Iwatani, Tomoyuki Miura, Tatsuhiko Igarashi, Hironori Sato and Akio Adachi : Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors., Journal of Virology, Vol.87, No.21, 11447-11461, 2013.
(Summary)
Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5 susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.
Akatsuki Saito, Masako Nomaguchi, Ken Kono, Yasumasa Iwatani, Masaru Yokoyama, Yasuhiro Yasutomi, Hironori Sato, Tatsuo Shioda, Wataru Sugiura, Tetsuro Matano, Akio Adachi, E Emi Nakayama and Hirofumi Akari : TRIM5 genotypes in cynomolgus monkeys primarily influence inter-individual diversity in susceptibility to monkey-tropic human immunodeficiency virus type 1., The Journal of General Virology, Vol.94, No.Pt 6, 1318-1324, 2013.
(Summary)
TRIM5 restricts human immunodeficiency virus type 1 (HIV-1) infection in cynomolgus monkey (CM) cells. We previously reported that a TRIMCyp allele expressing TRIM5-cyclophilin A fusion protein was frequently found in CMs. Here, we examined the influence of TRIM5 gene variation on the susceptibility of CMs to a monkey-tropic HIV-1 derivative (HIV-1mt) and found that TRIMCyp homozygotes were highly susceptible to HIV-1mt not only in vitro but also in vivo. These results provide important insights into the inter-individual differences in susceptibility of macaques to HIV-1mt.
Masako Nomaguchi, Naoya Doi, Sachi Fujiwara, Akatsuki Saito, Hirofumi Akari, E Emi Nakayama, Tatsuo Shioda, Masaru Yokoyama, Hironori Sato and Akio Adachi : Systemic biological analysis of the mutations in two distinct HIV-1mt genomes occurred during replication in macaque cells., Microbes and Infection, Vol.15, No.4, 319-328, 2013.
(Summary)
Fundamental property of viruses is to rapidly adapt themselves under changing conditions of virus replication. Using HIV-1 derivatives that poorly replicate in macaque cells as model viruses, we studied here mechanisms for promoting viral replication in non-natural host cells. We found that the HIV-1s could evolve to grow better in both macaque and human cells by the continuous culture in macaque lymphocyte cell lines. Notably, only several mutations at defined sites of the Pol-integrase and/or the Env-gp120 reproducibly appeared in repeated adaptation experiments and were sufficient to cause the phenotypic change. Meanwhile, no amino acid changes to enhance viral replication in macaque cells were found in interaction sites for the known anti-retroviral proteins. These findings disclose a hitherto unappreciated evolutionary pathway to augment HIV-1 replication in primate cells, where tuning of viral interactions with positive rather than negative factors for replication can play a dominant role.
Masako Nomaguchi, Masaru Yokoyama, Ken Kono, E Emi Nakayama, Tatsuo Shioda, Akatsuki Saito, Hirofumi Akari, Yasuhiro Yasutomi, Tetsuro Matano, Hironori Sato and Akio Adachi : Gag-CA Q110D mutation elicits TRIM5-independent enhancement of HIV-1mt replication in macaque cells., Microbes and Infection, Vol.15, No.1, 56-65, 2013.
(Summary)
HIV-1 is strictly adapted to humans, and cause disease-inducing persistent infection only in humans. We have generated a series of macaque-tropic HIV-1 (HIV-1mt) to establish non-human primate models for basic and clinical studies. HIV-1mt clones available to date grow poorly in macaque cells relative to SIVmac239. In this study, viral adaptive mutation in macaque cells, G114E in capsid (CA) helix 6 of HIV-1mt, that enhances viral replication was identified. Computer-assisted structural analysis predicted that another Q110D mutation in CA helix 6 would also increase viral growth potential. A new proviral construct MN4Rh-3 carrying CA-Q110D exhibited exquisitely enhanced growth property specifically in macaque cells. Susceptibility of MN4Rh-3 to macaque TRIM5/TRIMCyp proteins was examined by their expression systems. HIV-1mt clones so far constructed already completely evaded TRIMCyp restriction, and further enhancement of TRIMCyp resistance by Q110D was not observed. In addition, Q110D did not contribute to evasion from TRIM5 restriction. However, the single-cycle infectivity of MN4Rh-3 in macaque cells was enhanced relative to the other HIV-1mt clones. Our results here indicate that CA-Q110D accelerates viral growth in macaque cells irrelevant to TRIM5 proteins restriction.
Kei Miyakawa, Tatsuya Sawasaki, Satoko Matsunaga, Andrey Tokarev, Gary Quinn, Hirokazu Kimura, Masako Nomaguchi, Akio Adachi, Naoki Yamamoto, John Guatelli and Akihide Ryo : Interferon-induced SCYL2 limits release of HIV-1 by triggering PP2A-mediated dephosphorylation of the viral protein Vpu., Science Signaling, Vol.5, No.245: ra73, 2012.
(Summary)
Human cells respond to infection by retroviruses through the actions of proteins that inhibit the spread of viruses to other cells. One example is bone marrow stromal cell antigen 2 (BST2; also known as tetherin), which is an interferon (IFN)-inducible protein that restricts the release of progeny virions from infected cells. The HIV-1 accessory protein Vpu (viral protein U) causes degradation of BST2, and phosphorylation of Vpu at residues Ser(52) and Ser(56) is required for this function. We report that the host protein SCY1-like protein 2 (SCYL2) mediates the dephosphorylation of Vpu, antagonizing Vpu function and facilitating BST2-dependent restriction of HIV-1 release. SCYL2 reduced the number of virus particles released from cells infected with wild-type HIV-1, but not a strain lacking vpu, in a BST2-dependent manner. SCYL2 stimulated the dephosphorylation of Vpu on Ser(52) and Ser(56) by recruiting protein phosphatase 2A (PP2A) to Vpu. Conversely, depletion of SCYL2 resulted in enhanced phosphorylation of Vpu and increased viral particle release. Moreover, SCYL2 was produced in response to type I IFN and contributed to IFN-mediated viral restriction. Together, these results suggest that SCYL2 serves as a regulatory factor for Vpu, reducing the extent of Vpu phosphorylation and consequently enhancing BST2-mediated viral restriction.
(Keyword)
Clathrin / HIV-1 / Human Immunodeficiency Virus Proteins / Humans / Interferons / Phosphorylation / Protein Binding / Protein Phosphatase 2 / Protein-Serine-Threonine Kinases / Viral Regulatory and Accessory Proteins / Virion
Mikako Fujita, Masako Nomaguchi, Akio Adachi and Masami Otsuka : SAMHD1-Dependent and -Independent Functions of HIV-2/SIV Vpx Protein., Frontiers in Microbiology, Vol.3, No.297.doi:10.3389/fmicb.2012.00297., 2012.
(Summary)
Both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) encode a unique set of accessory proteins that enhance viral replication in the host. Two similar accessory proteins, Vpx and Vpr, are encoded by HIV-2. In contrast, HIV-1 encodes Vpr but not Vpx. Recent studies have indicated that Vpx counteracts a particular host restriction factor, thereby facilitating reverse transcription in myeloid cells such as monocyte-derived macrophages and monocyte-derived dendritic cells. This mechanism of counteraction is similar to that of the accessory proteins Vif and Vpu which antagonize other host factors. In 2011, the protein SAMHD1 was identified as the restriction factor counteracted by Vpx. Studies have since revealed that SAMHD1 degrades deoxynucleoside triphosphates (dNTPs), which are components of viral genomic cDNA, in order to deprive viruses of dNTPs. Although interactions between SAMHD1 and Vpx continue to be a major research focus, Vpx has also been shown to have an apparent ability to enhance nuclear import of the viral genome in T lymphocytes. This review summarizes the current knowledge regarding SAMHD1-dependent and -independent functions of Vpx, and discusses possible reasons why HIV-2 encodes both Vpx and Vpr, unlike HIV-1.
Masako Nomaguchi, Naoya Doi, Yui Matsumoto, Yosuke Sakai, Sachi Fujiwara and Akio Adachi : Species tropism of HIV-1 modulated by viral accessory proteins., Frontiers in Microbiology, Vol.3, No.267.doi:10.3389/fmicb.2012.00267., 2012.
(Summary)
Human immunodeficiency virus type 1 (HIV-1) is tropic and pathogenic only for humans, and does not replicate in macaque monkeys routinely used for experimental infections. This specially narrow host range (species tropism) has impeded much the progress of HIV-1/acquired immunodeficiency syndrome (AIDS) basic research. Extensive studies on the underlying mechanism have revealed that Vif, one of viral accessory proteins, is critical for the HIV-1 species tropism in addition to Gag-capsid protein. Another auxiliary protein Vpu also has been demonstrated to affect this HIV-1 property. In this review, we focus on functional interactions of these HIV-1 proteins and species specific-restriction factors. In addition, we describe an evolutional viewpoint that is relevant to the species tropism of HIV-1 controlled by the accessory proteins.
Akatsuki Saito, Ken Kono, Masako Nomaguchi, Yasuhiro Yasutomi, Akio Adachi, Tatsuo Shioda, Hirofumi Akari and E Emi Nakayama : Geographical, genetic and functional diversity of antiretroviral host factor TRIMCyp in cynomolgus macaque (Macaca fascicularis)., The Journal of General Virology, Vol.93, No.Pt 3, 594-602, 2012.
(Summary)
The antiretroviral factor tripartite motif protein 5 (TRIM5) gene-derived isoform (TRIMCyp) has been found in at least three species of Old World monkey: rhesus (Macaca mulatta), pig-tailed (Macaca nemestrina) and cynomolgus (Macaca fascicularis) macaques. Although the frequency of TRIMCyp has been well studied in rhesus and pig-tailed macaques, the frequency and prevalence of TRIMCyp in cynomolgus macaques remain to be definitively elucidated. Here, the geographical and genetic diversity of TRIM5/TRIMCyp in cynomolgus macaques was studied in comparison with their anti-lentiviral activity. It was found that the frequency of TRIMCyp in a population in the Philippines was significantly higher than those in Indonesian and Malaysian populations. Major and minor haplotypes of cynomolgus macaque TRIMCyp with single nucleotide polymorphisms in the cyclophilin A domain were also found. The functional significance of the polymorphism in TRIMCyp was examined, and it was demonstrated that the major haplotype of TRIMCyp suppressed human immunodeficiency virus type 1 (HIV-1) but not HIV-2, whilst the minor haplotype of TRIMCyp suppressed HIV-2 but not HIV-1. The major haplotype of TRIMCyp did not restrict a monkey-tropic HIV-1 clone, NL-DT5R, which contains a capsid with the simian immunodeficiency virus-derived loop between -helices 4 and 5 and the entire vif gene. These results indicate that polymorphisms of TRIMCyp affect its anti-lentiviral activity. Overall, the results of this study will help our understanding of the genetic background of cynomolgus macaque TRIMCyp, as well as the host factors composing species barriers of primate lentiviruses.
(Keyword)
Animals / Cyclophilin A / Genetic Variation / HIV-1 / HIV-2 / Haplotypes / Indonesia / Macaca fascicularis / Malaysia / Molecular Sequence Data / Philippines / Phylogeography / Protein Isoforms / Sequence Analysis, DNA
Yasuyuki Miyazaki, Ariko Miyake, Masako Nomaguchi and Akio Adachi : Structural dynamics of retroviral genome and the packaging., Frontiers in Microbiology, Vol.2, No.264.doi:10.3389/fmicb.2011.00264, 2011.
(Summary)
Retroviruses can cause diseases such as AIDS, leukemia, and tumors, but are also used as vectors for human gene therapy. All retroviruses, except foamy viruses, package two copies of unspliced genomic RNA into their progeny viruses. Understanding the molecular mechanisms of retroviral genome packaging will aid the design of new anti-retroviral drugs targeting the packaging process and improve the efficacy of retroviral vectors. Retroviral genomes have to be specifically recognized by the cognate nucleocapsid domain of the Gag polyprotein from among an excess of cellular and spliced viral mRNA. Extensive virological and structural studies have revealed how retroviral genomic RNA is selectively packaged into the viral particles. The genomic area responsible for the packaging is generally located in the 5' untranslated region (5' UTR), and contains dimerization site(s). Recent studies have shown that retroviral genome packaging is modulated by structural changes of RNA at the 5' UTR accompanied by the dimerization. In this review, we focus on three representative retroviruses, Moloney murine leukemia virus, human immunodeficiency virus type 1 and 2, and describe the molecular mechanism of retroviral genome packaging.
Nopporn Chutiwitoonchai, Masateru Hiyoshi, Philip Mwimanzi, Takamasa Ueno, Akio Adachi, Hirotaka Ode, Hironori Sato, T Oliver Fackler, Seiji Okada and Shinya Suzu : The identification of a small molecule compound that reduces HIV-1 Nef-mediated viral infectivity enhancement., PLoS ONE, Vol.6, No.11, e27696, 2011.
(Summary)
Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of viral production itself. However, the infectivity of the viruses produced in the presence of 2c was significantly lower than that of control viruses. Importantly, an inhibitory effect was observed with Nef(+) wild-type viruses, but not with viruses produced in the absence of Nef or in the presence of proline-rich PxxP motif-disrupted Nef, both of which displayed significantly reduced intrinsic infectivity. Meanwhile, the overexpression of the SH3 domain of the tyrosine kinase Hck, which binds to a PxxP motif in Nef, also reduced viral infectivity. Importantly, 2c inhibited Hck SH3-Nef binding, which was more marked when Nef was pre-incubated with 2c prior to its incubation with Hck, indicating that both Hck SH3 and 2c directly bind to Nef and that their binding sites overlap. These results imply that both 2c and the Hck SH3 domain inhibit the interaction of Nef with an unidentified host protein and thereby reduce Nef-mediated infectivity enhancement. The first inhibitory compound 2c is therefore a valuable chemical probe for revealing the underlying molecular mechanism by which Nef enhances the infectivity of HIV-1.
Shun Adachi, Akio Adachi and Masako Nomaguchi : Commentary on a New Era of Investigating 3D Structure-Based Human-Virus Protein Network Dynamics., Frontiers in Microbiology, Vol.2, No.186.doi:10.3389/fmicb.2011.00186., 2011.
Masako Nomaguchi, Mikako Fujita and Akio Adachi : The Fourth Major Restriction Factor Against HIV/SIV., Frontiers in Microbiology, Vol.2, No.132.doi:10.3389/fmicb.2011.00132., 2011.
The viral protein Nef is a key element for the progression of HIV disease. Previous in vitro studies suggested that Nef expression in T-cell lines enhanced TCR signaling pathways upon stimulation with TCR cross-linking, leading to the proposal that Nef lowers the threshold of T-cell activation, thus increasing susceptibility to viral replication in immune response. Likewise, the in vivo effects of Nef transgenic mouse models supported T-cell hyperresponse by Nef. However, the interpretation is complicated by Nef expression early in the development of T cells in these animal models. Here, we analyzed the consequence of Nef expression in ovalbumin-specific/CD4(+) peripheral T cells by using a novel mouse model and demonstrate that Nef inhibits antigen-specific T-cell proliferation and multiple functions required for immune response in vivo, which includes T-cell helper activity for the primary and memory B-cell response. However, Nef does not completely abrogate T-cell activity, as defined by low levels of cytokine production, which may afford the virus a replicative advantage. These results support a model, in which Nef expression does not cause T-cell hyperresponse in immune reaction, but instead reduces the T-cell activity, that may contribute to a low level of virus spread without viral cytopathic effects.
Masako Nomaguchi and Akio Adachi : HIV-1 Vpr and G2 cell cycle arrest., Future Microbiology, Vol.6, No.4, 375-378, 2011.
(Summary)
Evaluation of: Belzile J-P, Abrahamyan LG, Gerard FCA et al.: Formation of mobile chromatin-associated nuclear foci containing HIV-1 Vpr and VPRBP is critical for the induction of G2 cell cycle arrest. PLoS Pathog. 6(9), E1001080 (2010). All primate immunodeficiency viruses encode a unique set of accessory proteins to optimize their replication in hosts. In general, these proteins appear to be multifunctional for virus replication. Viral protein R (Vpr), one of the accessory proteins, has also been reported to exhibit distinct activities, but its exact role in the viral life cycle is still unclear and controversial. However, of particular note, Vpr-mediated G2 cell cycle arrest is conserved among primate immunodeficiency viruses. Belzile et al. have characterized and analyzed in detail the punctuate structures on the DNA of host cells formed by HIV-1 Vpr (Vpr nuclear foci). They demonstrate, mainly by confocal immunofluorescence analysis, that highly mobile chromatin-associated Vpr nuclear foci are critical for induction of the G2 cell cycle arrest.
Masako Nomaguchi, N. Doi, S. Fujiwara, M. Fujita and Akio Adachi : Site-defective mutants with distinct ability to down-modulate cell surface CD4 and tetherin, Frontiers in Microbiology, Vol.1, No.116, 2010.
(Summary)
HIV-1 Vpu acts positively on viral infectivity by mediating CD4 degradation in endoplasmic reticulum and enhances virion release by counteracting a virion release restriction factor, tetherin. In order to define the impact of Vpu activity on HIV-1 replication, we have generated a series of site-specific proviral vpu mutants. Of fifteen mutants examined, seven exhibited a replication-defect similar to that of a vpu-deletion mutant in a lymphocyte cell line H9. These mutations clustered in narrow regions within transmembrane domain (TMD) and cytoplasmic domain (CTD). Replication-defective mutants displayed the reduced ability to enhance virion release from a monolayer cell line HEp2 without exception. Upon transfection with Vpu expression vectors, neither TMD mutants nor CTD mutants blocked CD4 expression at the cell surface in another monolayer cell line MAGI. While TMD mutants were unable to down-modulate cell surface tetherin in HEp2 cells, CTD mutants did quite efficiently. Confocal microscopy analysis revealed the difference of intracellular localization between TMD and CTD mutants. In total, replication capability of HIV-1 carrying vpu mutations correlates well with the ability of Vpu to enhance virion release and to impede the cell surface expression of CD4 but not with the ability to down-modulate cell surface tetherin. Our results here suggest that efficient viral replication requires not only down-regulation of cell surface tetherin but also its degradation.
Akatsuki Saito, Masako Nomaguchi, Sayuki Iijima, Ayumu Kuroishi, Tomoyuki Yoshida, Young-Jung Lee, Toshiyuki Hayakawa, Ken Kono, Emi E. Nakayama, Tatsuo Shioda, Yasuhiro Yasutomi, Akio Adachi, Tetsuro Matano and Hirofumi Akari : Improved capacity of a monkey-tropic HIV-1 derivative to replicate in cynomolgus monkeys with minimal modifications., Microbes and Infection, Vol.13, No.1, 58-64, 2010.
(Summary)
Human immunodeficiency virus type 1 (HIV-1) hardly replicates in Old World monkeys. Recently, a mutant HIV-1 clone, NL-DT5R, in which a small part of gag and the entire vif gene are replaced with SIVmac239-derived ones, was shown to be able to replicate in pigtail monkeys but not in rhesus monkeys (RM). In the present study, we found that a modified monkey-tropic HIV-1 (HIV-1mt), MN4-5S, acquired the ability to replicate efficiently in cynomolgus monkeys as compared with the NL-DT5R, while neither NL-DT5R nor MN4-5S replicated in RM cells. These results suggest that multiple determinants may be involved in the restriction of HIV-1 replication in macaques, depending on the species of macaques. The new HIV-1mt clone will be useful for studying molecular mechanisms by which anti-viral host factors regulate HIV-1 replication in macaques.
Naoya Doi, Sachi Fujiwara, Akio Adachi and Masako Nomaguchi : Growth ability in various macaque cell lines of HIV-1 with simian cell-tropism., The Journal of Medical Investigation : JMI, Vol.57, No.3-4, 284-292, 2010.
(Summary)
We have recently constructed a series of novel human immunodeficiency viruses (HIV-1s) that are tropic for a macaque cell line (mt; macaque cell-tropic) to generate and establish a primate experimental system for HIV-1/AIDS study. In order to determine biological properties of these viruses effectively, several other macaque cell lines with distinct characteristics that can be routinely and easily used, instead of primary cells, for infection experiments are required. In this study, we have examined four macaque cell lines for their surface expression of virus receptor molecules and for their genotype of a major anti-viral capsid gene. Furthermore, we monitored the susceptibility of the cell lines to a standard simian immunodeficiency virus (SIV) clone and three representative basic mt HIV-1 clones. Results obtained here have clearly indicated that these cell lines are exquisitely useful to characterize various SIVs and more importantly, mt HIV-1s.
Masako Nomaguchi and Akio Adachi : Virology as biosystematics: towards understanding the viral infection biology, Frontiers in Microbiology, Vol.1, No.2, 2010.
Tamiko Nagao, Tomoki Yamashita, Ariko Miyake, Tsuneo Uchiyama, Masako Nomaguchi and Akio Adachi : Different interaction between HIV-1 Vif and its cellular target proteins APOBEC3G/APOBEC3F., The Journal of Medical Investigation : JMI, Vol.57, No.1-2, 89-94, 2010.
(Summary)
We examined a series of site-directed point mutants of human immunodeficiency virus type 1 (HIV-1) Vif for their interaction with cellular anti-viral factors APOBEC3G/APOBEC3F. Mutant viruses that display growth-defect in H9 cells did not counteract effectively APOBEC3G and/or APOBEC3F without exception, as monitored by single-cycle infectivity assays. While growth-defective mutants of Vif C-terminal region were unable to suppress APOBEC3G/APOBEC3F, some N-terminal region mutants did neutralize one of APOBEC3G/APOBEC3F. These data have suggested that members of APOBEC3 family other than APOBEC3G/APOBEC3F are not important for anti-HIV-1 activity. Furthermore, APOPEC3G/APOBEC3F were found to differently associate with Vif in virions as analyzed by equilibrium density centrifugation. Taken together, these results indicated that interaction of HIV-1 Vif and APOBEC3G is distinct from that between Vif and APOBEC3F.
(Keyword)
Cell Line / Cytidine Deaminase / Cytosine Deaminase / Humans / vif Gene Products, Human Immunodeficiency Virus
Mikako Fujita, Masami Otsuka, Masako Nomaguchi and Akio Adachi : Multifaceted activity of HIV Vpr/Vpx proteins: the current view of their virological functions., Reviews in Medical Virology, Vol.20, No.2, 68-76, 2010.
(Summary)
Primate immunodeficiency viruses encode viral proteins that are uniquely auxiliary to their growth in host cells. Of these accessory proteins, those designated Vpr and Vpx are least well understood with respect to their functions in the viral replication cycle. Moreover, their assigned roles based on the results in published studies remain controversial. This review summarises current knowledge on human immunodeficiency virus (HIV) Vpr/Vpx proteins, and discusses their functional activities during the viral life cycle in macrophages and T lymphocytes, the two major target cells of HIV infection.
(Keyword)
CD4-Positive T-Lymphocytes / HIV / Humans / Macrophages / Viral Regulatory and Accessory Proteins / vpr Gene Products, Human Immunodeficiency Virus
Tomoki Yamashita, Masako Nomaguchi, Ariko Miyake, Tsuneo Uchiyama and Akio Adachi : Status of APOBEC3G/F in cells and progeny virions modulated by Vif determines HIV-1 infectivity., Microbes and Infection, Vol.12, No.2, 166-171, 2009.
(Summary)
We examined various HIV-1 Vif mutants for interaction with APOBEC3 proteins (A3G/A3F). All replication-defective proviral mutants were found to carry A3G/A3F in virions, and of these, a replication-defective mutant with Vif that binds to A3G in cells but not in virions was noted. Furthermore, a mutant Vif protein that suppresses A3F activity but does not exclude A3F from virions was identified. We also showed that incorporation of Vif into virions is dependent on its interaction with A3G/A3F. Taken together, we concluded that functional binding of Vif to A3G/A3F in cells and/or virions is critical for viral infectivity.
Abhay Jere, Mikako Fujita, Akio Adachi and Masako Nomaguchi : Role of HIV-1 Nef protein for virus replication in vitro., Microbes and Infection, Vol.12, No.1, 65-70, 2009.
(Summary)
The Nef protein of primate lentiviruses (simian and human immunodeficiency viruses; SIV/HIVs) appears to be multi-functional and plays a pivotal role in viral persistence and pathogenesis in vivo. Of its numerous functions reported to date, the ability to enhance virion infectivity in indicator cell lines and to augment viral replication in peripheral blood mononuclear cells (PBMCs) and lymphocytes (PBLs) is very well conserved among various SIV/HIVs. This review summarizes and organizes current knowledge of HIV-1 Nef with respect to this particularly virological activity for understanding the basis of its in vivo function.
Ayumu Kuroishi, Akatsuki Saito, Yasuhiro Shingai, Tatsuo Shioda, Masako Nomaguchi, Akio Adachi, Hirofumi Akari and Emi E. Nakayama : Modification of a loop sequence between alpha-helices 6 and 7 of virus capsid (CA) protein in a human immunodeficiency virus type 1 (HIV-1) derivative that has simian immunodeficiency virus (SIVmac239) vif and CA alpha-helices 4 and 5 loop improves replication in cynomolgus monkey cells., Retrovirology, Vol.6, 70, 2009.
(Summary)
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees but not cynomolgus or rhesus monkeys while simian immunodeficiency virus isolated from macaque (SIVmac) readily establishes infection in those monkeys. Several HIV-1 and SIVmac chimeric viruses have been constructed in order to develop an animal model for HIV-1 infection. Construction of an HIV-1 derivative which contains sequences of a SIVmac239 loop between alpha-helices 4 and 5 (L4/5) of capsid protein (CA) and the entire SIVmac239 vif gene was previously reported. Although this chimeric virus could grow in cynomolgus monkey cells, it did so much more slowly than did SIVmac. It was also reported that intrinsic TRIM5alpha restricts the post-entry step of HIV-1 replication in rhesus and cynomolgus monkey cells, and we previously demonstrated that a single amino acid in a loop between alpha-helices 6 and 7 (L6/7) of HIV type 2 (HIV-2) CA determines the susceptibility of HIV-2 to cynomolgus monkey TRIM5alpha. RESULTS: In the study presented here, we replaced L6/7 of HIV-1 CA in addition to L4/5 and vif with the corresponding segments of SIVmac. The resultant HIV-1 derivatives showed enhanced replication capability in established T cell lines as well as in CD8+ cell-depleted primary peripheral blood mononuclear cells from cynomolgus monkey. Compared with the wild type HIV-1 particles, the viral particles produced from a chimeric HIV-1 genome with those two SIVmac loops were less able to saturate the intrinsic restriction in rhesus monkey cells. CONCLUSION: We have succeeded in making the replication of simian-tropic HIV-1 in cynomolgus monkey cells more efficient by introducing into HIV-1 the L6/7 CA loop from SIVmac. It would be of interest to determine whether HIV-1 derivatives with SIVmac CA L4/5 and L6/7 can establish infection of cynomolgus monkeys in vivo.
Tamiko Nagao, Kazuki Hatcho, Naoya Doi, Sachi Fujiwara, Akio Adachi and Masako Nomaguchi : Amino acid alterations in Gag that confer the ability to grow in simian cells on HIV-1 are located at a narrow CA region., The Journal of Medical Investigation : JMI, Vol.56, No.1-2, 21-25, 2009.
(Summary)
We previously generated a prototype monkey-tropic human immunodeficiency virus type 1 (HIV-1) designated NL-DT5R. This viral clone has a small region of simian immunodeficiency virus (SIV) within Gag capsid (CA) protein and also SIV Vif protein, but displays a poor growth phenotype in simian cells. To improve the growth potential of NL-DT5R, we have constructed a series of its gag variant viruses. Out of fourteen viral clones generated, five were infectious for simian HSC-F cells, and two of the infectious variants grew similarly with NL-DT5R. Taking their genome structures into consideration, our data here clearly show that a narrow CA region within the Gag protein, i.e., the domain around cyclophilin A (CypA)-binding loop, is critical for the growth ability of HIV-1 in simian cells.
Kazuya Kamada, Tomoki Yamashita, Kazuki Hatcho, Akio Adachi and Masako Nomaguchi : Evasion from CypA- and APOBEC-mediated restrictions is insufficient for HIV-1 to efficiently grow in simian cells., Microbes and Infection, Vol.11, No.2, 164-171, 2008.
(Summary)
We have recently generated a monkey cell-tropic virus termed NL-DT5R from an HIV-1 NL4-3 clone and demonstrated that both cyclophilin A (CypA)-binding loop in Gag-capsid (CA) and Vif are responsible for the species-restriction of HIV-1. In this study, we constructed 16 CypA-binding loop mutants from the HIV-1-derivative NL-DT5R, and analyzed them biologically and biochemically. The mutants displayed various multi-cycle infection potencies in cynomolgus monkey (CyM) HSC-F cells, but none of them grew significantly better than NL-DT5R. Consistently, any of the HIV-1 variants examined here did not effectively counter CyM TRIM5alpha as judged by single-cycle infectivity assays. Assessment of their single-cycle infectivity in simian and CyM TRIM5alpha-expressing feline cells in the presence of cyclosporin A (CsA) showed that intervention of CypA-CA interaction did not restore full NL-DT5R infectivity, while CsA increased infectivity of DT5R/4-3 carrying the sequence of NL4-3 CypA-binding loop up to the NL-DT5R level. Almost similar data were obtained in the experiments utilizing CypA-targeting siRNA. Together with our previous results regarding NL-DT5R, these data suggested that evasion from CypA- and APOBEC-mediated restrictions is still insufficient for HIV-1 to completely overcome the species barrier.
(Keyword)
Animals / Cats / Cell Line / Cercopithecus aethiops / Cyclophilin A / Cytidine Deaminase / HIV-1 / Humans / Macaca fascicularis / Molecular Sequence Data
Mikako Fujita, Masami Otsuka, Masako Nomaguchi and Akio Adachi : Functional region mapping of HIV-2 Vpx protein., Microbes and Infection, Vol.10, No.12-13, 1387-1392, 2008.
(Summary)
To determine functional regions of HIV-2 Vpx, we analyzed a series of site-specific vpx-mutants for their growth potentials in lymphocytic cells and compared the results with those in macrophages. We found that amino acid residues important for virus growth in lymphocytic cells, in macrophages, and in both are clustered separately in Vpx. Through generation and characterization of new vpx-mutants, we further demonstrated that a remarkable proline-stretch present at the C-terminus of Vpx is critical for its stable expression, thereby contributing to its functional activity. Taken together, there can be functionally distinct regions in HIV-2 Vpx.
Masako Nomaguchi, Mikako Fujita and Akio Adachi : Role of HIV-1 Vpu protein for virus spread and pathogenesis., Microbes and Infection, Vol.10, No.9, 960-967, 2008.
(Summary)
Vpu is an accessory viral protein almost unique to HIV-1 among primate immunodeficiency viruses, and has two major functions: degradation of the CD4 molecule in endoplasmic reticulum and enhancement of virion release from cells. Recent identification of a novel host restriction factor, tetherin, as a Vpu-antagonist suggests that Vpu contributes to virus spread by facilitating progeny virion production. This review focuses on the two distinct functions of Vpu and summarizes current knowledge on its virological role in the HIV-1 life cycle.
(Keyword)
Antigens, CD4 / HIV Infections / HIV-1 / Human Immunodeficiency Virus Proteins / Humans / Viral Regulatory and Accessory Proteins / Virion
Kazuki Hatcho, Kazuya Kamada, Tomoki Yamashita, Akio Adachi and Masako Nomaguchi : Replication potentials of vif variant viruses generated from monkey cell-tropic HIV-1 derivative clones NL-DT5/NL-DT5R., Microbes and Infection, Vol.10, No.10-11, 1218-1222, 2008.
(Summary)
To obtain monkey-tropic viruses that are more closely related to HIV-1 than the original NL-DT5/NL-DT5R clones, we constructed six vif-chimeric and two site-specific vif-mutant viruses, and examined their growth ability. Different from NL-DT5/NL-DT5R, these viruses did not grow in monkey cells. We monitored the capability of the mutants to antagonize monkey APOBEC3G/F by single-cycle infectivity assays. They counteracted poorly or not at all the action of the APOBEC3G/F. Our results have indicated that the native SIVmac Vif is required to overcome the species barrier against HIV-1.
Tomoki Yamashita, Kazuya Kamada, Kazuki Hatcho, Akio Adachi and Masako Nomaguchi : Identification of amino acid residues in HIV-1 Vif critical for binding and exclusion of APOBEC3G/F., Microbes and Infection, Vol.10, No.10-11, 1142-1149, 2008.
(Summary)
To define a region(s) in human immunodeficiency virus type 1 (HIV-1) Vif that involves binding to its target APOBEC3G (A3G), we have generated a series of site-specific proviral vif mutants. Of 30 mutants examined, 15 did not grow at all or grew more poorly than wild-type virus in non-permissive cells. Eight clones with N-terminal mutations located outside of the HCCH motif and BC-box, which are known to be directly crucial for the degradation of A3G, were chosen from these growth-defective mutants and mainly analyzed in detail for functional activity of their mutant Vif proteins. By single-cycle replication and immunoprecipitation/immunoblotting analyses, mutants designated W21A, S32A, W38A, Y40A, and H43A were demonstrated to hardly or poorly bind to and neutralize A3G. Upon transfection, these mutants produced progeny virions containing much more A3G than wild-type clone. Interestingly, while mutants designated E76A and W79A acted normally to inactivate A3G, they were found to exhibit a Vif-defective phenotype against A3F. Another unique mutant designated Y69A incompetent against both of A3G/F was also identified. Our results here have indicated that at least two distinct regions in the N-terminal half of HIV-1 Vif are critical for binding and exclusion of A3G/F.
Mikako Fujita, Masami Otsuka, Masami Miyoshi, Boonruang Khamsri, Masako Nomaguchi and Akio Adachi : Vpx is critical for reverse transcription of the human immunodeficiency virus type 2 genome in macrophages., Journal of Virology, Vol.82, No.15, 7752-7756, 2008.
(Summary)
The abilities of wild-type and vpx-defective human immunodeficiency virus type 2 (HIV-2) clones to synthesize viral DNA in human monocyte-derived macrophages (MDMs) and lymphocytic cells were comparatively and quantitatively evaluated. While the vpx-defective mutant directed the synthesis of viral DNA comparably to the wild-type virus and normally in lymphocytic cells, no appreciable viral DNA was detected in MDMs infected with the mutant. To substantiate this finding and to determine whether there is some specific region(s) in Vpx crucial for viral DNA synthesis in MDMs, we generated a series of site-specific point mutants of vpx and examined their phenotypes. The resultant five mutants, with no infectivity for MDMs, showed, without exception, the same defect as the vpx-defective mutant. Our results here clearly demonstrated that the entire Vpx protein is critical for reverse transcription of the HIV-2 genome in human MDMs.
Masako Nomaguchi, Naoya Doi, Kazuya Kamada and Akio Adachi : Species barrier of HIV-1 and its jumping by virus engineering., Reviews in Medical Virology, Vol.18, No.4, 261-275, 2008.
(Summary)
Monkey infection models are absolutely necessary for studies of human immunodeficiency virus type 1 (HIV-1) pathogenesis and of developing drugs/vaccines against HIV-1. In addition, currently unknown roles of its accessory proteins for in vivo replication await elucidation by experimental approaches. Due to the fact that HIV-1 is tropic only for chimpanzees and humans, studies of this line have been impeded for a long time, although various investigations have been carried out utilising genetically related SIV and SIV/HIV chimeric virus (SHIV) as pathogens. Recent findings of anti-HIV-1 innate factors such as tripartite motif protein 5alpha (TRIM5alpha) and APOBEC3G/F prompted us to re-initiate an old and vital research project which would, as a result, confer the capability to overcome the species barrier on the HIV-1. We currently have obtained, by virus engineering through genetic manipulation and adaptation, some new and promising HIV-1 clones for in vivo studies in macaque monkeys as mentioned above. In this review, we summarise the past, present and future of HIV-1/SIV chimeric viruses with special reference to relevant basic HIV-1/SIV studies.
Tomoki Yamashita, Naoya Doi, Akio Adachi and Masako Nomaguchi : Growth ability in simian cells of monkey cell-tropic HIV-1 is greatly affected by downstream region of the vif gene., The Journal of Medical Investigation : JMI, Vol.55, No.3-4, 236-240, 2008.
(Summary)
To obtain monkey-tropic (mt) HIV-1 derivatives with distinct biological characteristics and to improve the viral growth property, we have generated several variants from a prototype mt HIV-1 designated NL-DT5R (X4-tropic). The prototype HIV-1 contains a portion of gag and entire vif genes from SIVmac in its genome. The two derivatives carrying 3' half-genomic region of the SF162 (R5-tropic) or 89.6 (dual-tropic) isolate displayed very retarded or no viral growth, respectively, in a simian cell line HSC-F. In contrast, the three clones containing a part of env gene (encoding the V1-V4 region) from SF162, YU-2 (R5-tropic) or 89.6 showed different growth kinetics in HSC-F cells, although they grew somewhat more poorly than the NL-DT5R. Comparison of various viral proteins potentially involved in the different biological properties has revealed that, while amino acid sequences of Tat, Rev, Vpr, Vpu and Nef are quite conserved among the clones, those in the surface (SU) region of Env are relatively heterologous. Our data described here have shown that the 3' half of viral genome other than gag and vif genes greatly affects the growth property of mt HIV-1 in simian cells.
Tatsuhiko Igarashi, Ranjini Lyengar, Russel A. Byrum, Alicia Buckler-White, Robin L. Dewar, Charles E. Buckler, H. Clifford Lane, Kazuya Kamada, Akio Adachi and Malcolm A. Martin : Human immunodeficiency virus type 1 derivative with 7% simian immunodeficiency virus genetic content is able to establish infections in pig-tailed macaques, Journal of Virology, Vol.81, No.20, 11549-11552, 2007.
(Summary)
A human immunodeficiency virus type 1 (HIV-1) derivative (HIV(NL-DT5R)) containing sequences encoding a 7-amino-acid segment of CA and the entire vif gene from simian immunodeficiency virus (SIV) was previously shown to establish spreading infections in cultured macaque peripheral blood mononuclear cells. To assess its replicative and disease-inducing properties in vivo, HIV(NL-DT5R) was inoculated into pig-tailed macaques. HIV(NL-DT5R) generated plasma viremia in all five of the monkeys and elicited humoral responses against all of the HIV-1 structural proteins but did not cause CD4(+) T-lymphocyte depletion or clinical disease. Additional adaptation will be required to optimize infectivity in vivo.
Ahmad Piroozmand, Yoshihiko Yamamoto, Boonruang Khamsri, Mikako Fujita, Tsuneo Uchiyama and Akio Adachi : Generation and characterization of APOBEC3G-positive 293T cells for HIV-1 Vif study, The Journal of Medical Investigation : JMI, Vol.54, No.1-2, 154-158, 2007.
(Summary)
We have established a number of 293T cell lines that express a human anti HIV-1 factor APOBEC3G. Out of seven cell clones examined, four were readily demonstrated to express APOBEC3G by immunoblotting analysis. In particular, two clones (A3G-C1 and -C4) were found to produce a much higher level of functional APOBEC3G relative to that by pooled cell clones. The transfection efficiency of all these cell clones were similar to that of the parental cells, producing a comparable level of virions upon transfection of wild type and vif-minus proviral DNA clones. Furthermore, the expression level of APOBEC3G in the best cell line (A3G-C1) was far much higher than those of an APOBEC3G-positive lymphocyte cell line and peripheral blood mononuclear cells. We finally monitored the incorporation of APOBEC3G into virions produced in A3G-C1. APOBEC3G was easily detected in progeny viral particles upon transfection of vif-minus proviral clone but not of wild type. These results indicated that our new A3G-C1 cell line is eminently useful for various studies on the interaction of human APOBEC3G and HIV-1 Vif.
Kazuya Kamada, Tatsuhiko Igarashi, Malcolm A. Martin, Boonruang Khamsri, Kazuki Hatcho, Tomoki Yamashita, Mikako Fujita, Tsuneo Uchiyama and Akio Adachi : Generation of HIV-1 derivatives that productively infect macaque monkey lymphoid cells, Proceedings of the National Academy of Sciences of the United States of America, Vol.103, No.45, 16959-16964, 2006.
(Summary)
The narrow host range of human immunodeficiency virus type 1 (HIV-1) is caused in part by innate cellular factors such as apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) and TRIM5alpha, which restrict virus replication in monkey cells. Variant HIV-1 molecular clones containing both a 21-nucleotide simian immunodeficiency virus (SIV) Gag CA element, corresponding to the HIV-1 cyclophilin A-binding site, and the entire SIV vif gene were constructed. Long-term passage in a cynomolgus monkey lymphoid cell line resulted in the acquisition of two nonsynonymous changes in env, which conferred improved replication properties. A proviral molecular clone, derived from infected cells and designated NL-DT5R, was used to generate virus stocks capable of establishing spreading infections in the cynomolgus monkey T cell line and CD8-depleted peripheral blood mononuclear cells from five of five pig-tailed macaques and one of three rhesus monkeys. NL-DT5R, which genetically is >93% HIV-1, provides the opportunity, not possible with currently available SIV/HIV chimeric viruses, to analyze the function of multiple HIV-1 genes in a broad range of nonhuman primate species.
B Khamsri, M. Fujita, K. Kamada, A. Piroozmand, T. Yamasahita, T. Uchiyama and Akio Adachi : Effects of lysine to arginine mutations in HIV-1 Vif on its expression and viral infectivity, International Journal of Molecular Medicine, Vol.18, No.4, 679-683, 2006.
(Summary)
We previously demonstrated that the expression in cells of human immunodeficiency virus type 1 (HIV-1) Vif is maintained at low level by proteasome-degradation. We examined the contribution of 16 lysines present in Vif (NL432 clone), which is composed of 192 amino acids (aa), to its expression within cells and to viral infectivity for non-permissive cells. To this end, various lysine-arginine mutations were introduced into wild-type (wt) Vif, and the mutational effects were monitored by transfection experiments. When all the lysines were changed to arginines, the mutant Vif was expressed in cells at much higher level than wt and was much more stable. Both N-terminal (aa nos. 34 and 36) and C-terminal (aa nos. 179 and 181) lysines were found to be almost sufficient for wt property. Different from this observation, one of the lysines at aa nos. 22 and 26 was demonstrated to be essential for the virus to grow in non-permissive cells. Our results showed that there is no clear co-relationship between the expression level of HIV-1 Vif and viral infectivity.
A. Piroozmand, B. Khamsri, M. Fujita, Akio Adachi and T. Uchiyama : Morphological study on biologically distinct vpx/vpr mutants of HIV-2, The Journal of Medical Investigation : JMI, Vol.53, No.3-4, 271-276, 2006.
(Summary)
We have previously shown that human immunodeficiency virus type 2 (HIV-2) without functional vpx and vpr genes is severely defective for viral growth in lymphocytic cells, and suggested that the virions produced in the absence of Vpx and Vpr are critically damaged. To examine the nature of replication-defect for the vpx/vpr double mutant, we quantitatively and morphologically studied the virions produced in cells transfected or infected with wild type clone, single (vpx and vpr mutants) or the double mutant. While no significant difference in virion production was found for various virus clones in transfected cells, a major growth retardation in infected cells was readily observed for the vpx and vpx/vpr mutants. In particular, no viral growth was detected for the double mutant. By contrast to the very distinct growth characteristics of the three mutant clones, no appreciable difference in virion morphology was noted. These results indicated that Vpx and Vpr of HIV-2 may cooperatively contribute to virion infectivity without affecting virion morphogenesis.
K Kamada, Akiko Yoshida, Boonruang Khamsri, A Piroozmand, T Yamashita, Tsuneo Uchiyama, Mikako Fujita and Akio Adachi : Construction of gag-chimeric viruses between HIV-1 and SIVmac that are capable of productive multi-cycle infection, Microbes and Infection, Vol.8, No.4, 1075-1081, 2006.
(Summary)
Forty-nine recombinant viral clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus from the rhesus monkey (SIVmac), which carry chimeric gag (capsid/p2 region) genes in the background of the HIV-1 genome, were constructed to establish an HIV-1/monkey infection model system for human AIDS. Upon transfection, all the recombinants generated progeny virions at a level comparable to the parental HIV-1 clone and no major abnormalities were found in the virions, as examined by Western blot analysis. In infection experiments, 18 recombinants grew in human lymphocytic cells and six of these clones propagated as well as the parental virus, as monitored by virion associated-reverse transcriptase production. By contrast, none of the recombinants grew at a detectable level in monkey lymphocytic cells. The defective replication site(s) in human cells for non-infectious recombinants was mapped to the step before and/or during reverse transcription. Our results described here showed that HIV-1 type chimeric viruses between HIV-1 and SIVmac, which are capable of spreading productive infection, are readily constructed throughout the capsid/p2 region. In addition, it is suggested that there may be a viral determinant(s), other than Gag, responsible for the species-specific tropism of HIV-1 and which is associated with viral DNA synthesis.
Boonruang Khamsri, Fumiko Murao, Akiko Yoshida, Akiko Sakurai, Tsuneo Uchiyama, Hiroki Shirai, Yo Matsuo, Mikako Fujita and Akio Adachi : Comparative study on the structure and cytopathogenic activity of HIV Vpr/Vpx proteins, Microbes and Infection, Vol.8, No.1, 10-15, 2006.
(Summary)
The three-dimensional (3-D) structure of human immunodeficiency virus type 2 (HIV-2) Vpr/Vpx was predicted by homology modeling based on the NMR structure of human immunodeficiency virus type 1 (HIV-1) Vpr. The three proteins similarly have three major amphipathic alpha-helices. In contrast to HIV-1 Vpr, Vpr/Vpx of HIV-2 have a long N-terminal loop and clustered prolines in the second half of the C-terminal loop. HIV-2 Vpx uniquely contains a long region between the second and third major helices, and bears several glycines in the first half of the C-terminal loop. Instead of the glycines, there is a group of hydrophilic amino acids and arginines in the corresponding regions of the two Vprs. To compare the cytopathogenic potentials of HIV-1 Vpr and HIV-2 Vpr/Vpx, we examined the production of luciferase as a marker of cell damage. We further analyzed the characteristics of cells transduced with vpr/vpx genes driven by an inducible promoter. The results obtained clearly show that structurally similar, but distinct, HIV Vpr/Vpx proteins are detrimental to target cells.
(Keyword)
Amino Acid Sequence / Cytopathogenic Effect, Viral / Gene Expression Regulation / Gene Products, vpr / HIV-1 / HIV-2 / HeLa Cells / Humans / Molecular Sequence Data / Protein Conformation / Sequence Alignment / Viral Regulatory and Accessory Proteins / vpr Gene Products, Human Immunodeficiency Virus
Akiko Yoshida, Ahmad Piroozmand, Akiko Sakurai, Mikako Fujita, Tsuneo Uchiyama, Tooru Kimura, Yoshio Hayashi, Yoshiaki Kiso and Akio Adachi : Establishment of a biological assay system for human retroviral protease activity, Microbes and Infection, Vol.7, No.5-6, 820-824, 2005.
(Summary)
In order to obtain indicator cell lines that are exquisitely susceptible to human T-lymphotropic virus type 1 (HTLV-1), luciferase gene driven by HTLV-1 long terminal repeat (LTR) was transfected into lymphocytic H9 cells with neo gene, and cell lines were selected by G418. A cell line (H9/K30luc) was found to produce an extremely high level of luciferase only when co-cultured with HTLV-1 producer MT-2 cells. Both in the absence and presence of a reverse transcriptase (RT) inhibitor azidothymidine, H9/K30luc cells generated similarly high luciferase activity upon co-cultivation with MT-2 cells. To develop an equivalent system for human immunodeficiency virus type 1 (HIV-1), H9/NL432 cells, which are stably infected with HIV-1 and producing a low level of the virus-like MT-2 cells for HTLV-1, were generated. Together with the indicator cell line H9/H1luc for HIV-1 already reported, antiviral effects of some agents on HTLV-1 and HIV-1 could be readily and sensitively evaluated by similar methods. In fact, by using our system, an HIV-1 protease inhibitor, saquinavir, was demonstrated to be highly effective against HIV-1 but not against HTLV-1.
Huaqing Wang, Akiko Sakurai, Boonruang Khamsri, Tsuneo Uchiyama, Hongxi Gu, Akio Adachi and Mikako Fujita : Unique characteristics of HIV-1 Vif expression, Microbes and Infection, Vol.7, No.3, 385-390, 2005.
(Summary)
We examined the steady-state expression in cells of four accessory proteins of human immunodeficiency virus type 1 (HIV-1). For this purpose, a series of single gene expression vectors for these viral proteins were constructed and were monitored for their production by transfection. Among them, the expression level of Vif was found to be lowest in both the absence and presence of APOBEC3G. In addition, we noticed the presence of its truncated form, which was not observed for the other accessory proteins. When a subgenomic vector was used for transfection, authentic and several small forms of Vif were produced. By mutational analysis, these forms were demonstrated to be mutant Vif proteins translated from M8, M16 and M29. When a full-length molecular clone was used, the smaller versions of Vif were hardly observed. Functional analysis of these mutant Vif proteins showed that they are incapable of modulating viral infectivity. The results described above, i.e. the low steady-state expression and the presence of truncated forms, represent the unique characteristics of HIV-1 Vif.
Abhay Jere, Ahmad Piroozmand, Spikanth Tripathy, Ramesh Paranjape, Akiko Sakurai, Mikako Fujita and Akio Adachi : Generation and characterization of HIV-1 clones chimeric for subtypes B and C nef, International Journal of Molecular Medicine, Vol.14, No.6, 1087-1090, 2004.
(Summary)
The impact of human immunodeficiency virus type 1 (HIV-1) Nef on viral infectivity was evaluated by characterization of chimeric clones. Chimera with respect to the nef gene were constructed between subtypes B and C, and monitored for their replication in human peripheral blood mononuclear cells. The parental clones used were pNL432 (subtype B) and pIndie-C1 (Indian subtype C), which show considerable sequence heterogeneity in nef and distinct viral growth phenotype. While an enhancing effect of Nef on viral infectivity was noted, no significant growth difference was observed between the parental and chimeric clones. The difference in growth potential of the two subtype clones was mainly ascribable to viral sequence(s) other than nef. Our results here clearly showed that HIV-1 Nef does not significantly affect the in vitro viral infectivity in natural target cells.
(Keyword)
human immunodeficiency virus type 1 / subtype B / subtype C / Nef
Tamiko Nagao, Akiko Yoshida, Akiko Sakurai, Ahmad Piroozmand, Abhay Jere, Mikako Fujita, Tsuneo Uchiyama and Akio Adachi : Determination of HIV-1 infectivity by lymphocytic cell lines with integrated luciferase gene, International Journal of Molecular Medicine, Vol.14, No.6, 1073-1076, 2004.
(Summary)
We have established lymphocytic cell lines H9 and M8166 that contain integrated copy of luciferase gene under the control of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). While H9 is known to be non-permissive for or insensitive to some particular mutant strains of HIV/simian immunodeficiency virus (SIV), M8166 is one of the most susceptible lines to various HIV/SIVs. The luciferase gene driven by HIV-1 LTR was transfected into H9 and M8166 cells with the neo gene, and cell lines were selected by G418. The indicator cell lines thus obtained were designated H9/H1luc and M8166/H1luc, and monitored for their susceptibility to various HIV clones including in vitro-constructed mutants. Both cell lines, particularly M8166/H1luc, were found to be exquisitely sensitive to HIV-1 and HIV-2. Furthermore, they exhibited the response to infections by various viral clones exactly as expected from the characteristics of the original cell lines. These results indicated that our new indicator cell lines H9/H1luc and M8166/H1luc are eminently useful for a variety of molecular virological studies on HIV/SIV.
Ahmad Piroozmand, Hajime A Koyama, Yoshiko Shimada, Mikako Fujita, Tsutomu Arakawa and Akio Adachi : Role of Us3 gene of herpes simplex virus type 1 for resistance to interferon, International Journal of Molecular Medicine, Vol.14, No.4, 641-645, 2004.
(Summary)
The sensitivity of a Us3-deletion mutant virus of herpes simplex virus type 1 (HSV-1) to consensus interferon (IFN) was compared with that of parental wild-type (wt) and the repaired viruses. Although one-step growth of the Us3-deficient virus in the IFN-treated HEp-2 cells was not markedly affected at a high multiplicity of infection (MOI), both the progeny virus yield and cytopathic effect were suppressed in a significantly higher degree in the mutant virus-infected cells than those in wt or the repaired virus-infected cells. This enhanced IFN-sensitivity of the mutant virus was more clearly demonstrated by the infection at a low MOI. In addition, both the size and number of plaques of the Us3-deficient virus in Vero cells were remarkably reduced with increasing concentrations of IFN, compared to those of wt or the repaired virus. These results indicate that the deletion of Us3 gene makes HSV-1 more sensitive to IFN.
Fumihiro Sugahara, Tsuneo Uchiyama, Hitoshi Watanabe, Yukie Shimazu, Masaru Kuwayama, Yutaka Fujii, Katsuhiro Kiyotani, Akio Adachi, Nobuoki Kohno, Tetsuya Yoshida and Takemasa Sakaguchi : Paramyxovirus Sendai virus-like particle formation by expression of multiple viral proteins and acceleration of its release by C protein., Virology, Vol.325, No.1, 1-10, 2004.
(Summary)
Envelope viruses maturate by macromolecule assembly and budding. To investigate these steps, we generated virus-like particles (VLPs) by co-expression of structural proteins of Sendai virus (SeV), a prototype of the family Paramyxoviridae. Simultaneous expression of matrix (M), nucleo- (N), fusion (F), and hemagglutinin-neuraminidase (HN) proteins resulted in the generation of VLPs that had morphology and density similar to those of authentic virus particles, although the efficiency of release from cells was significantly lower than that of the virus. By using this VLP formation as a model of virus budding, roles of individual proteins in budding were investigated. The M protein was a driving force of budding, and the F protein facilitated and the HN protein suppressed VLP release. Either of the glycoproteins, F or HN, as well as the N protein, significantly shifted density of VLPs to that of virus particles, suggesting that viral proteins bring about integrity of VLPs by protein-protein interactions. We further found that co-expression of a nonstructural protein, C, but not V, enhanced VLP release to a level comparable to that of virus particles, demonstrating that the C protein plays a role in virus budding.
(Keyword)
Sendai virus / Virus-like particle / Morphology / Density / Nucleocapsid / C protein
Akiko Sakurai, Abhay Jere, Akiko Yoshida, Takeshi Yamada, Aikichi Iwamoto, Akio Adachi and Mikako Fujita : Functional analysis of HIV-1 vif genes derived from Japanese long-term nonprogressors and progressors for AIDS, Microbes and Infection, Vol.6, No.9, 799-805, 2004.
(Summary)
We analyzed the function of human immunodeficiency virus type 1 (HIV-1) vif gene from Japanese long-term nonprogressors (LTNPRs) and progressors (PRs) for acquired immunodeficiency syndrome (AIDS). We constructed a basic HIV-1 infectious clone, which facilitated the incorporation and evaluation of vif from infected individuals. Proviral reporter clones carrying vif from six Japanese LTNPRs and seven PRs were then generated and their in vitro growth kinetics were analyzed. The vif clones, which could confer infectivity on reporter viruses, were considered active, and the ratio of the active clones to the number of clones examined per individual was determined. For the majority of LTNPRs, there was no correlation between presence or absence of functional vif with long-term nonprogression for AIDS. There was one exception in which all the clones examined had inactive vif, suggesting a probable association of inactive vif with the nonprogression. All PRs with high viral load had a high ratio of active vif clones. Our results suggest that the presence of functional vif would influence HIV-1 infectivity and disease progression in infected individuals.
Mikako Fujita, Hirofumi Akari, Akiko Sakurai, Akiko Yoshida, Tomoki Chiba, Keiji Tanaka, Klaus Strebel and Akio Adachi : Expression of HIV-1 accessory protein Vif is controlled uniquely to be low and optimal by proteasome degradation, Microbes and Infection, Vol.6, No.9, 791-798, 2004.
Hirofumi Akari, Mikako Fujita, Sandra Kao, Mohammad A. Khan, Miranda Shehu-Xhilaga, Akio Adachi and Klaus Strebel : High Level Expression of Human Immunodeficiency Virus Type-1 Vif Inhibits Viral Infectivity by Modulating Proteolytic Processing of the Gag Precursor at the p2/Nucleocapsid Processing Site, The Journal of Biological Chemistry, Vol.279, No.13, 12355-12362, 2004.
(Summary)
The human immunodeficiency virus type-1 Vif protein has a crucial role in regulating viral infectivity. However, we found that newly synthesized Vif is rapidly degraded by cellular proteases. We tested the dose dependence of Vif in non-permissive H9 cells and found that Vif, when expressed at low levels, increased virus infectivity in a dose-dependent manner. Surprisingly, however, the range of Vif required for optimal virus infectivity was narrow, and further increases in Vif severely reduced viral infectivity. Inhibition of viral infectivity at higher levels of Vif was cell type-independent and was associated with an accumulation of Gag-processing intermediates. Vif did not act as a general protease inhibitor but selectively inhibited Gag processing at the capsid and nucleocapsid (NC) boundary. Identification of Vif variants that were efficiently packaged but were unable to modulate Gag processing suggests that Vif packaging was necessary but insufficient for the production of 33- and 34-kDa processing intermediates. Interestingly, these processing intermediates, like Vif, associated with viral nucleoprotein complexes more rigidly than mature capsid and NC. We conclude that virus-associated Vif inhibits processing of a subset of Gag precursor molecules at the p2/NC primary cleavage site. Modulation of processing of a small subset of Gag molecules by physiological levels of Vif may be important for virus maturation. However, the accumulation of such processing intermediates at high levels of Vif is inhibitory. Thus, rapid intracellular degradation of Vif may have evolved as a mechanism to prevent such inhibitory effects of Vif.
Hajime A Koyama, Akio Adachi and Hiroshi Irie : Physiological significance of apoptosis during animal virus infection, International Reviews of Immunology, Vol.22, No.5-6, 341-359, 2003.
(Summary)
Apoptosis has been considered to be a host defense mechanism against viral infection in multicellular organisms. This is based on the findings that apoptogenic mutants of insect viruses cannot grow because infected host cells die by apoptosis. This suggests that the apoptotic response of host cells has a deleterious effect on virus infection. Thus, apoptosis is an important host defense mechanism that is capable of inhibiting viral replication during infection. However, in vitro studies indicated that apoptosis alone does not provide the same protection against viral infection in animal cells as it does in the insect cells. Still, most animal viruses have acquired a strategy to overcome host cell apoptosis. In addition, a varying degree of necrosis usually accompanies apoptosis, suggesting a possible contribution of necrosis to the host reactions against virus. To understand the physiological significance of apoptosis during animal virus infection, we have characterized viral growth and the cellular responses against virus infection in a wide variety of virus-cell interaction systems. Mainly based on our own works, we discuss the nature of apoptosis in the animal virus infection and verify its role as a host defense mechanism against virus infection.
Mikako Fujita, Akiko Yoshida, Akiko Sakurai, Junko Tatsuki, Fumiko Ueno, Hirofumi Akari and Akio Adachi : Susceptibility of HVS-immortalized lymphocytic HSC-F cells to various strains and mutants of HIV/SIV, International Journal of Molecular Medicine, Vol.11, No.5, 641-644, 2003.
(Summary)
Susceptibility of HSC-F, a cynomolgus macaque cell line immortalized by Herpesvirus saimiri, to infection with various primate immunodeficiency viruses were monitored. While NL432 clone of human immunodeficiency virus type 1 (HIV-1) did not grow at all in HSC-F cells, GH123 and GL-AN clones of HIV-2, and MA239 clone of simian immunodeficiency virus isolated from macaque monkeys (SIVMAC) did grow in these cells. In addition, NM-3 clone of a chimeric simian and human immunodeficiency virus (SHIV) grew fairly well in HSC-F cells. Mutational analyses of accessory genes of GL-AN were successfully performed in the HSC-F cells. These results have thus demonstrated the importance of this cell line for molecular biological studies on HIV/SIV.
Fumiko Ueno, Hiroshi Shiota, Maki Miyaura, Akiko Yoshida, Akiko Sakurai, Junko Tatsuki, Hajime A Koyama, Hirofumi Akari, Akio Adachi and Mikako Fujita : Vpx and Vpr proteins of HIV-2 up-regulate the viral infectivity by a distinct mechanism in lymphocytic cells, Microbes and Infection, Vol.5, No.5, 387-395, 2003.
(Summary)
Mutants of human immunodeficiency virus type 2 (HIV-2) carrying a frame-shift mutation in vpx, vpr, and in both genes were monitored for their growth potentials in a newly established lymphocytic cell line, HSC-F. Worthy of note, the replication of a vpx single mutant, but not vpr, was severely impaired in these cells, and that of a vpx-vpr double mutant was more damaged. Defective replication sites of the vpx single and vpx-vpr double mutants were demonstrated to be mapped, respectively, to the nuclear import of viral genome, and to both, this process and the virus assembly/release stage. While the mutational effect of vpr was small, the replication efficiency in one cycle of the vpx mutant relative to that of wild-type virus was estimated to be 10%. The growth phenotypes of the vpx, vpr, and vpx-vpr mutant viruses in HSC-F cells were essentially repeated in primary human lymphocytes. In primary human macrophages, whereas the vpx and vpx-vpr mutants did not grow at all, the vpr mutant grew equally as well as the wild-type virus. These results strongly suggested that Vpx is critical for up-regulation of HIV-2 replication in natural target cells by enhancing the genome nuclear import, and that Vpr promotes HIV-2 replication somewhat, at least in lymphocytic cells, at a very late replication phase.
Hajime A Koyama, Hiroshi Irie, Atsushi Kato, Yoshiyuki Nagai and Akio Adachi : Virus multiplication and induction of apoptosis by Sendai virus: role of the C proteins, Microbes and Infection, Vol.5, No.5, 373-378, 2003.
(Summary)
Sendai virus (SeV) P gene encodes a nested set of carboxyl-coterminal proteins (C', C, Y1 and Y2), which are referred to collectively as the C proteins. Characterization of the virus multiplication and cellular responses in HEp-2 cells infected with the recombinant SeV which lacks two (C' and C), three (C', C and Y1) or all the four C proteins revealed that all the recombinant viruses can grow in the cells to various extents, depending, apparently, on the number of species expressing C protein. In reverse proportion to the viral growth ability, these viruses induced apoptosis in the infected cells. These results indicate that Y2 protein has an antiapoptotic activity, and suggest that this activity works in an additive manner with the longer C protein(s) (C' and/or C) of SeV in order to suppress virus-induced apoptosis in the SeV-infected cells. Apparently, the antiapoptotic activity of the C proteins supports virus multiplication in the infected cells.
(Keyword)
Sendai virus / apoptosis / Antiapoptosis / C proteins
Masataka Nishimura, Michiyuki Maeda, Jun-ichiro Yasunaga, Hideshi Kawakami, Ryuji Kaji, Akio Adachi, Takashi Uchiyama and Masao Matsuoka : Influence of cytokine and mannose binding protein gene polymorphisms on human t-cell leukemia virus type i (hTLV-i) provirus load in HTLV-I asymptomatic carriers, Human Immunology, Vol.64, No.4, 453-457, 2003.
(Summary)
Human T-cell leukemia virus type I (HTLV-I) provirus load differs more than 100-fold among carriers and a high provirus load in the peripheral blood mononuclear cells (PBMCs) is regarded as a risk factor for both preleukemic states and inflammatory diseases including HTLV-I-associated myelopathy (HAM). We examined polymorphisms in the genes for tumor necrosis factor (TNF), TNF receptor type 1 and 2, lymphotoxin (LT)-alpha, interleukin (IL)-1beta, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, and mannose binding protein (ManBP) in 143 HTLV-I carriers whether these polymorphisms affect the provirus load in the PBMCs of carriers. No significant association was observed between these polymorphisms and the provirus load. Homozygotes for a ManBP-variant allele, however, showed a tendency for the decreased number of provirus load. When combined, the data on the alleles of LT-alpha and MCP-1, HTLV-I carriers having high producer alleles of both genes showed a trend for increased provirus load. These data suggest that inflammation or an active immune response may induce an increased amount of HTLV-I-infected T cells, leading to a high provirus load.
(Keyword)
lymphotoxin (LT)-α / monocyte chemoattractant protein (MCP)-1 / mannose binding protein (ManBP) / human T-cell leukemia virus type I (HTLV-I) / HTLV-I associated myelopathy (HAM)
Mikako Fujita, Akiko Sakurai, Akiko Yoshida, Maki Miyaura, Hajime A Koyama, Koji Sakai and Akio Adachi : Amino Acid Residues 88 and 89 in the Central Hydrophilic Region of Human Immunodeficiency Virus Type 1 Vif Are Critical for Viral Infectivity by Enhancing the Steady-State Expression of Vif, Journal of Virology, Vol.77, No.2, 1626-1632, 2003.
(Summary)
A hydrophilic region consisting of strikingly clustered charged amino acids is present at the center of human immunodeficiency virus type 1 (HIV-1) Vif. In this study, the role for this central hydrophilic region (E(88)WRKKR(93)) in the virus replication in nonpermissive H9 cells was investigated by extensive deletion and substitution analysis. A total of 31 mutants were constructed. Deletion of the E(88) or W(89) residue alone abolished viral infectivity in H9 cells and impaired virus replication in primary macrophage cultures. Substitution analysis indicated that the hydrophilicity and charge of the central region are insignificant for the function of Vif. Of the 16 substitution mutants, 3 mutants with substitution of E(88) and W(89) with an A residue did not grow in H9 cells. Upon transfection, four mutants (i.e., two mutants with deletion of E(88) or W(89); a mutant with substitution of E(88) and W(89) with A; and a mutant with substitution of E(88), W(89), and R(90) with A) were found to express Vif at a very reduced level relative to that by the wild-type clone. These results have thus demonstrated that amino acid residues 88 and 89 of Vif are critical for the replication of HIV-1 in target cells by enhancing the steady-state expression of Vif. In addition, E(88) and W(89) residues were found to be extremely conserved among the Vif proteins of naturally occurring HIV-1 field isolates as well as those of laboratory HIV-1 strains.
Mikako Fujita, Sora Matsumoto, Akiko Sakurai, Naoya Doi, Maki Miyaura, Akiko Yoshida and Akio Adachi : Apparent lack of trans-dominant negative effects of various vif mutants on the replication of HIV-1, Microbes and Infection, Vol.4, No.12, 1203-1207, 2002.
(Summary)
The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for virus growth in non-permissive cells such as H9. To elucidate the mechanism of action of the Vif protein, vif mutants, which show trans-dominant negative effects on the replication of HIV-1, would be useful tools. In this study, a new assay system to identify the mutants of this category was established. For this new system, various reporter clones carrying both mutant and authentic vif sequences were generated. By determining the growth ability of the viruses derived from the reporter constructs, the potential negative effect of the mutant vif sequence was readily and sensitively monitored. Ten vif mutant sequences tested were found not to exert the trans-dominant negative effect on the replication of HIV-1.
Masataka Nishimura, Masao Matsuoka, Michiyuki Maeda, Ikuko Mizut, Shuji Mita, Makoto Uchino, Makoto Matsui, Yasuo Kuroda, Hideshi Kawakami, Ryuji Kaji, Akio Adachi and Takashi Uchiyama : Association between interleukin-6 gene polymorphism and human T-Cell leukemia virus type I associated myelopathy, Human Immunology, Vol.63, No.8, 696-700, 2002.
(Summary)
We studied cytokine gene polymorphisms in the promoter region, including interleukin (IL)-6, IL-1beta, and IL-10, in Japanese patients with human T-cell leukemia virus type I (HTLV-I) associated myelopathy (HAM) (n = 65), asymptomatic HTLV-I carriers (n = 143), and HTLV-I seronegative, normal controls (n = 160). There was a significant difference between HAM patients and HTLV-I carriers in the distribution of IL-6 promoter polymorphism at position -634 (chi(2) = 9.90, p = 0.0071). The IL-6 genotype was also significantly different between HAM patients and normal controls (chi(2) = 11.53, p = 0.0033), while a similar distribution was observed in IL-1beta and IL-10 polymorphisms among HAM patients, carriers, and normal controls. The results suggest that IL-6 gene region may contribute to susceptibility to HAM, and that aberrant cytokine productions could be involved in the development of HAM.
(Keyword)
IL-6 / IL-10 / IL-1β / human T-cell leukemia virus type I (HTLV-I) / HAM
Hiroko Tomiyama, Hirofumi Akari, Akio Adachi and Masafumi Takiguchi : Different Effects of Nef-Mediated HLA Class I Down-Regulation on Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Cytolytic Activity and Cytokine Production, Journal of Virology, Vol.76, No.15, 7535-7543, 2002.
(Summary)
A previous study using a Nef-defective human immunodeficiency virus type 1 (HIV-1) mutant suggested that Nef-mediated down-regulation of HLA class I on the infected cell surface affects the cytolytic activity of HIV-1-specific cytotoxic T-lymphocyte (CTL) clones for HIV-1-infected primary CD4(+) T cells. We confirmed this effect by using a nef-mutant HIV-1 strain (NL-M20A) that expresses a Nef protein which does not induce down-regulation of HLA class I molecules but is otherwise functional. HIV-1-specific CTL clones were not able to kill primary CD4(+) T cells infected with a Nef-positive HIV-1 strain (NL-432) but efficiently lysed CD4(+) T cells infected with NL-M20A. Interestingly, CTL clones stimulated with NL-432-infected CD4(+) T cells were able to produce cytokines, albeit at a lower level than when stimulated with NL-M20A-infected CD4(+) T cells. This indicates that Nef-mediated HLA class I down-regulation affects CTL cytokine production to a lesser extent than cytolytic activity. Replication of NL-432 was partially suppressed in a coculture of HIV-1-infected CD4(+) T cells and HIV-1-specific CTL clones, while replication of NL-M20A was completely suppressed. These results suggest that HIV-1-specific CD8(+) T cells are able to partially suppress the replication of HIV-1 through production of soluble HIV-1-suppressive factors such as chemokines and gamma interferon. These findings may account for the mechanism whereby HIV-1-specific CD8(+) T cells are able to partially but not completely control HIV-1 replication in vivo.
Mikako Fujita, Akiko Sakurai, Akiko Yoshida, Sora Matsumoto, Maki Miyaura and Akio Adachi : Subtle mutations in the cysteine region of HIV-1 Vif drastically alter the viral replication phenotype, Microbes and Infection, Vol.4, No.6, 621-624, 2002.
(Summary)
Mutations were introduced into the region encoding the two cysteine and nearby amino acid residues of human immunodeficiency virus type 1 (HIV-1) Vif protein and, 12 single-amino-acid viral mutants were constructed. Determination of their growth characteristics in two lymphocytic cell lines revealed that only a single amino acid change in the cysteine region greatly altered the replication phenotype. In particular, the four mutants of amino acid 132 of Vif were grouped into three categories on the basis of their growth potentials. These results indicate that the cysteine region of Vif is critical for the cell-dependent replication efficiency of HIV-1.
(Keyword)
HIV-1 / Vif / Mutational analysis / Cysteine region
Hajime A Koyama, Hiroshi Irie, Fumiko Ueno, Motomi Ogawa, Akio Nomoto and Akio Adachi : Suppression of apoptotic and necrotic cell death by poliovirus, The Journal of General Virology, Vol.82, No.Pt 12, 2965-2972, 2001.
(Summary)
To determine an antiapoptotic activity of poliovirus type 1 (PV-1), we examined the effect of PV-1 infection on apoptosis that was induced in HEp-2 cells by the treatment with 1 M sorbitol. The virus did not induce apoptosis in the infected cells and could suppress both the fragmentation of chromosomal DNA and morphological cell and cell nuclei changes in the sorbitol-treated cells, indicating that PV-1 induces an antiapoptotic state. Comparison of the kinetics showed that this ability of the virus appeared in the infected cells at the time of progeny virus formation (maturation step of virus multiplication). Simultaneously with this antiapoptotic activity, PV-1 infection also suppressed non-apoptotic cell death induced by sodium chloride. Electron microscopic observation revealed that the cells killed by the sodium chloride treatment had undergone liquefactive necrosis, indicating that PV-1 can inhibit both apoptosis and necrosis. In addition, PV-1 can grow in the apoptotic cells, although the virus yield was reduced to a quarter of the yield in normal cells.
Akio Adachi, Maki Miyaura, Akiko Sakurai, Akiko Yoshida, Hajime A Koyama and Mikako Fujita : Growth characteristics of SHIV without the vpu gene, International Journal of Molecular Medicine, Vol.8, No.6, 641-644, 2001.
(Summary)
NM-3 is a prototype of chimeric virus between simian and human immunodeficiency viruses (SHIV). It grows in monkey lymphocytic cells in vitro and in vivo as well as in human cells. A mutant designated NM3-E65, which lacks expression of the entire vpu gene, was constructed from NM-3, and monitored for its replication property. Examination of growth properties in simian and human cells of SHIV, HIV, and their vpu mutants revealed that the vpu gene in the genome of the chimeric virus is functional, but non-essential for virus replication.
Hirofumi Akari, Stephan Bour, Sandra Kao, Akio Adachi and Klaus Strebel : The Human Immunodeficiency Virus Type 1 Accessory Protein Vpu Induces Apoptosis by Suppressing the Nuclear Factor B dependent Expression of Antiapoptotic Factors, The Journal of Experimental Medicine, Vol.194, No.9, 1299-1311, 2001.
(Summary)
Human immunodeficiency virus (HIV) type 1 Vpu is an integral membrane protein with a unique affinity for betaTrCP (TrCP), a key member of the SkpI-Cullin-F-box E3 ubiquitin ligase complex that is involved in the regulated degradation of cellular proteins, including IkappaB. Remarkably, Vpu is resistant to TrCP-mediated degradation and competitively inhibits TrCP-dependent degradation of IkappaB, resulting in the suppression of nuclear factor (NF)-kappaB activity in Vpu-expressing cells. We now report that Vpu, through its interaction with TrCP, potently contributes to the induction of apoptosis in HIV-infected T cells. Vpu-induced apoptosis is specific and independent of other viral proteins. Mutation of a TrCP-binding motif in Vpu abolishes its apoptogenic property, demonstrating a close correlation between this property of Vpu and its ability to inhibit NF-kappaB activity. The involvement of NF-kappaB in Vpu-induced apoptosis is further supported by the finding that the levels of antiapoptotic factors Bcl-xL, A1/Bfl-1, and TNF receptor-associated factor (TRAF)1, all of which are expressed in an NF-kappaB-dependent manner, are reduced and, at the same time, levels of active caspase-3 are elevated. Thus, Vpu induces apoptosis through activation of the caspase pathway by way of inhibiting the NF-kappaB-dependent expression of antiapoptotic genes.
Hajime A Koyama, Motomi Ogawa, Atsushi Kato, Yoshiyuki Nagai and Akio Adachi : Lack of apoptosis in Sendai virus-infected HEp-2 cells without participation of viral antiapoptosis gene, Microbes and Infection, Vol.3, No.13, 1115-1121, 2001.
(Summary)
Sendai virus (SeV) has been reported to induce apoptosis in many types of cells. In HEp-2 cells, however, it did not induce apoptosis in most of the infected cells under the conditions in which vesicular stomatitis virus induced massive apoptosis. The use of a novel technique, which allows the detection of viral antiapoptotic activity in the infected cells, showed that SeV does not have any antiapoptotic activity to interfere with the induction of apoptosis. Consistently, vesicular stomatitis virus-induced apoptosis was not interfered with by preinfection with SeV. These results indicate that the observed lack of apoptosis in these SeV-infected cells does not result from the suppression of apoptosis by viral antiapoptotic activity in the infected cells and suggest that, without activating a signaling pathway for the induction of apoptotic response in the infected cells, SeV can escape apoptosis of the cells, allowing long-term survival of the infected cells.
Mikako Fujita, Akiko Sakurai, Naoya Doi, Maki Miyaura, Akiko Yoshida, Koji Sakai and Akio Adachi : Analysis of the cell-dependent replication potentials of human immunodeficiency virus type 1 vif mutants, Microbes and Infection, Vol.3, No.13, 1093-1099, 2001.
(Summary)
Eleven in-frame vif gene mutants of HIV type 1 produced in non-permissive cells were examined for their replication potentials in various CD4-positive and -negative cell lines. Virus replication for each mutant was monitored by using several single- and multiple-cycle infectivity assays. Except for a mutant with wild-type phenotype, most mutants were severely defective for replication in all the cell lines as expected from the producer cell-dependent functioning of Vif so far reported. In contrast, two mutants, which have mutations in the hydrophilic or effector regions of Vif were found to have target cell-dependent replication potentials. These results demonstrate the presence of a novel category of the vif mutants important for elucidation of the Vif function.
Mikako Fujita, Akiko Yoshida, Maki Miyaura, Akiko Sakurai, Hirofumi Akari, Hajime A Koyama and Akio Adachi : Cyclophilin A-Independent Replication of a Human Immunodeficiency Virus Type 1 Isolate Carrying a Small Portion of the Simian Immunodeficiency Virus SIV(MAC) gag Capsid Region, Journal of Virology, Vol.75, No.21, 10527-10531, 2001.
(Summary)
Hybrid viruses between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus strain mac (SIV(MAC)) are invaluable to various fields of HIV-1 research. To date, however, no replication-competent HIV-1 strain containing the gag capsid (CA) region of SIV(MAC) has been reported. To obtain the viable gag gene chimeric virus in an HIV-1 background, seven HIV-1 strains carrying a part of SIV(MAC) CA or a small deletion in the CA region were constructed and examined for their biological and biochemical characteristics. While all the recombinants and mutants were found to express Gag and to produce progeny virions on transfection, only one chimeric virus, which has 18 bp of SIV gag CA sequence in place of the region encoding the HIV-1 CA cyclophilin A (CyPA)-binding loop, was infectious for human cell lines. Although this chimeric virus was unable to grow in monkey lymphocytic cells like wild-type (wt) HIV-1 did, it grew much better than wt virus in the presence of cyclosporin A in a human cell line which supports HIV-1 replication in a CyPA-dependent manner. These results indicate that the transfer of a small portion of the SIV(MAC) CA region to HIV-1 could confer the CyPA-independent replication potential of SIV(MAC) on the virus.
Y. Isaka, S. Miki, S. Kawauchi, A. Suyama, H. Sugimoto, Akio Adachi, T. Miura, M. Hayami, O. Yoshie, T. Fujiwara and A. Sato : A single amino acid change at Leu-188 in the reverse transcriptase of HIV-2 and SIV renders them sensitive to non-nucleoside reverse transcriptase inhibitors, Archives of Virology, Vol.146, No.4, 743-755, 2001.
(Summary)
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are selective for human immunodeficiency virus type 1 (HIV-1) and generally not effective on HIV-2 or simian immunodeficiency virus (SIV). Only SIVagm was found to be sensitive to NNRTIs. When the amino acid differences in RT between SIVmac and SIVagm were compared with the known amino acid substitutions of NNRTI-resistance variants of HIV-1, we came to consider that the amino acid residue Leu-188 of HIV-2 and SIVmac might be related to their resistance to NNRTIs. To test this hypothesis, we substituted Leu-188 to Cys or Tyr in HIV-2 and SIVmac, and examined sensitivity of the mutant molecular clones to NNRTIs. The L188Y mutant of HIV-2 became completely sensitive to delavirdine and efavirenz, while that of SIVmac was also significantly sensitive to these NNRTIs. We further isolated NNRTI-resistant variants from these mutant viruses and determined amino acid substitutions in RT. The roles of the observed substitutions in NNRTI-resistance were further confirmed by site-directed mutagenesis. Our study reveals the crucial role of L188 in the natural resistance of HIV-2 and SIVmac to NNRTIs. Furthermore, the observed substitutions in RT of HIV-2 and SIVmac support the common mechanism of action of NNRTIs against HIV-1, HIV-2 and SIV.
(Keyword)
Amino Acid Sequence / Amino Acid Substitution / Anti-HIV Agents / Benzoxazines / Cell Line / Delavirdine / Drug Resistance, Microbial / HIV Reverse Transcriptase / HIV-2 / HeLa Cells / Humans / Leucine / Molecular Sequence Data / Nevirapine / Oxazines / RNA-Directed DNA Polymerase / Reverse Transcriptase Inhibitors / Sequence Homology, Amino Acid / Simian immunodeficiency virus / Species Specificity
Y. Isaka, S. Miki, S. Kawauchi, A. Suyama, H. Sugimoto, Akio Adachi, M. Hayami, O. Yoshie, T. Fujiwara and A. Sato : Isolation and characterization of simian immunodeficiency virus variants that are resistant to nonnucleoside reverse transcriptase inhibitors, Archives of Virology, Vol.145, No.12, 2481-2492, 2000.
(Summary)
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) act quite specifically on human immunodeficiency virus type 1 (HIV-1). In general, they are not effective on human immunodeficiency virus type 2 (HIV-2) or simian immunodeficiency virus (SIV). Only SIV strains from African green monkeys are sensitive to several NNRTIs. Here we isolated NNRTI- and 3TC-resistant SIVagm variants. Viruses resistant to delavirdine contained V112I and M231I substitutions, while those resistant to 3TC contained a M 185I substitution. These amino acids are highly conserved in HIV-1, HIV-2, SIVmac and SIVagm, and the M184I (M185I in SIVagm) substitution was observed in 3TC-resistant HIV-1 and SIVmac. The roles of the observed mutations in NNRTI-resistance of SIVagm and HIV-1 were further confirmed by site-directed mutagenesis. The present results have provided a new insight into the common mechanism of sensitivity of HIV- 1 and SIVagm to NNRTIs.
Kazuya Hashinaka, Seiichi Hashida, Ichiro Nishikata, Akio Adachi, Shinichi Oka and Eiji Ishikawa : Recombinant p51 as Antigen in an Immune Complex Transfer Enzyme Immunoassay of Immunoglobulin G Antibody to Human Immunodeficiency Virus Type 1, Clinical and Diagnostic Laboratory Immunology, Vol.7, No.6, 967-976, 2000.
(Summary)
An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of Escherichia coli using a rather long DNA fragment (3,012 bp) of the whole HIV-1 pol gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced in E. coli as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to beta-D-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 using Ser-Ser-rp51 as antigen (Y) were well correlated to those obtained using rRT as antigen (X) (log Y = 0.99 log X + 0.23; r = 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1.
(Keyword)
Adult / Aged / Amino Acid Sequence / Antigen-Antibody Complex / Base Sequence / DNA Primers / Enzyme-Linked Immunosorbent Assay / Female / HIV Antibodies / HIV Antigens / HIV Reverse Transcriptase / HIV Seropositivity / HIV-1 / Humans / Immunoenzyme Techniques / Immunoglobulin G / Male / Middle Aged / Plasmids / Recombinant Proteins
Kyu-Bom Koh, Maki Miyaura, Akiko Yoshida, Akiko Sakurai, Mikako Fujita and Akio Adachi : Cell-dependent gag mutants of HIV-1 are crucially defective at the stage of uncoating/reverse transcription in non-permissive cells, Microbes and Infection, Vol.2, No.12, 1419-1423, 2000.
(Summary)
We have previously shown that some of the human immunodeficiency virus type 1 (HIV-1) gag matrix (MA), capsid (CA), and nucleocapsid (NC) mutants display host-cell-dependent replication potential, and that they are defective at the early phase of the virus replication cycle in non-permissive cells. To determine the defective replication stage of the cell-dependent mutants precisely, the processes of virus entry into cells and virus DNA synthesis were monitored by the highly sensitive enzyme-linked immunosorbent assay and polymerase chain reaction amplification analysis. The results obtained indicated that all the cell-dependent MA, CA and NC mutants are defective at the stage of uncoating/reverse transcription, and that a cellular factor(s) is involved in this process.
Maki Miyaura, Akiko Yoshida, Akiko Sakurai, Mikako Fujita, Hajime A Koyama and Akio Adachi : Mutational analysis of HIV-1 gag proteins, International Journal of Molecular Medicine, Vol.6, No.3, 265-269, 2000.
(Summary)
The mature Gag proteins of human immunodeficiency virus type 1 (HIV-1) are major components of infectious virions, and thought to carry out numerous functions throughout the HIV-1 replication cycle. We have recently generated numerous gag gene mutants of HIV-1 to genetically study the functions of the Gag proteins. Through the biological and biochemical analyses, our HIV-1 gag mutants have been grouped into early (defective for uncoating/reverse transcription), late (defective for virion release/maturation), and early/late (defective for both steps) mutants. Many mutants are found to efficiently inhibit the replication of wild-type virus. Worthy of note, there are some early mutants which show host cell-dependent replication potential.
Kazuya Hashinaka, Ichiro Nishikata, Seiichi Hashida, Akio Adachi, Shinichi Oka and Eiji Ishikawa : Preparations of recombinant HIV-1 p66 antigen to improve the specificity of immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 reverse transcriptase, Journal of Clinical Laboratory Analysis, Vol.14, No.4, 169-179, 2000.
(Summary)
Recombinant HIV-1 p66 (rp66, a subunit of reverse transcriptase (RT), a heterodimer of p66 and p51) was produced in Escherichia coli in three different ways. First, rp66 was produced as a part of the fusion protein of lacZ protein and HIV-1 pol protein consisting of three components: protease (p10), RT (p51/p66), and integrase (p31), and was released from the fusion protein by the protease (pol-rp66). Second, rp66 with Ser-Ser at the N-terminus was produced as a fusion protein with maltose-binding protein containing a factor Xa site between the two proteins (MBP-Ser-Ser-rp66) and was released from the fusion protein by factor Xa (Ser-Ser-rp66). Third, rp66 with Met-Gly at the N-terminus was produced in transformed cells (Met-Gly-rp66). The recombinant proteins were purified from sonic extracts of transformed cells by ammonium sulfate fractionation and various column chromatographies. MBP-Ser-Ser-rp66 and Met-Gly-rp66 were readily purified in sufficient amounts for labeling with 2, 4-dinitrophenyl groups and beta-D-galactosidase from E. coli, but pol-rp66 and Ser-Ser-rp66 were not for enzyme-labeling. Ser-Ser-rp66 was not only polymerized but also degraded to considerable extents. The purified preparations were labeled with 2,4-dinitrophenyl groups and beta-D-galactosidase and were tested in immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 RT using serum samples from 600 HIV-1 seronegative and 30 HIV-1 seropositive subjects. Among various combined uses of the two labeled preparations, the uses of 2,4-dinitrophenylated MBP-Ser-Ser-rp66 and pol-rp66 with beta-D-galactosidase-labeled Met-Gly-rp66 showed the highest (99.8%) and the second highest (99.5%) specificities, which were higher than that with the labeled preparations used in the previous study (98. 0%).
(Keyword)
reverse transcriptase / ecombinant protein / nzyme immunoassay / β-D-galactosidase / specificity / immunoglobulin G / HIV-1 infection / AIDS
Hajime A Koyama, Tomoharu Fukumori, Mikako Fujita, Hiroshi Irie and Akio Adachi : Physiological significance of apoptosis in animal virus infection, Microbes and Infection, Vol.2, No.9, 1111-1117, 2000.
(Summary)
In contrast to insect viruses, animal viruses can produce considerable amounts of progeny virus in cells undergoing apoptosis. Nevertheless, viruses in general have acquired the ability to escape apoptosis of infected cells. These facts indicate that the role of apoptosis in virus infection is different in insect virus and animal virus, although both viruses need to avoid apoptosis of the infected cells for a viral life cycle in nature. In animal virus infection, the primary role of apoptosis is considered not to be a premature lysis of the infected cells (and the following abortion of virus multiplication) but to allow the dying cells to be phagocytosed by macrophages. This phagocytosis is able to prevent dysregulated inflammatory reactions at the site of virus infection and to initiate a specific immune response against the infected virus.
Hirofumi Akari, Akiko Yoshida, Tomoharu Fukumori and Akio Adachi : Host cell-dependent replication of HIV-1 mutants with deletions in gp41 cytoplasmic tail region is independent of the function of Vif, Microbes and Infection, Vol.2, No.9, 1019-1023, 2000.
Akiko Yoshida, Maki Miyaura, Akiko Sakurai, Tomoharu Fukumori, Mikako Fujita, Hirofumi Akari and Akio Adachi : MHC-I expression in HTLV-1-positive and -negative cells, International Journal of Molecular Medicine, Vol.6, No.1, 83-86, 2000.
(Summary)
The expression level of major histocompatibility class I (MHC-I) and the extent of down-regulation of MHC-I after an anti-MHC-I antibody treatment in numerous human T-cell leukemia virus type 1 (HTLV-1)-positive and -negative lymphocytic cell lines were examined. While there was no clear correlation between the expression level of MHC-I and the presence of HTLV-1 genome, a relatively low level of MHC-I down-regulation was generally induced in HTLV-1-positive cells by the antibody. The results may suggest the potential involvement of MHC-I in HTLV-1 leukemogenesis.
(Keyword)
Antibodies, Monoclonal / Blotting, Western / Cell Line / Down-Regulation / Genes, MHC Class I / Histocompatibility Antigens Class I / Human T-lymphotropic virus 1 / Humans / Lymphocytes
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 10851271
Tomoharu Fukumori, Akari Hirofumi, Yoshida Akiko, Mikako Fujita, Koyama AH, Susumu Kagawa and Akio Adachi : Regulation of cell cycle and apoptosis by human immunodeficiency virus type 1 Vpr., Microbes and Infection, Vol.2, No.9, 1011-1017, 2000.
(Summary)
Biological effects of HIV-1 Vpr on CD4(+) cells were studied by an infection system. High-titered HIV-1 stocks pseudotyped with vesicular stomatitis virus G protein were prepared and used to inoculate into CD4(+ )T cells at high multiplicity of infection. Both cell- and virion-associated Vpr were demonstrated to arrest the cell cycle at the G2/M phase, and to induce cell apoptosis. Of note, morphologically apoptotic cells were shown to be arrested at the G2/M stage. No appreciable effect of Vpr on the anti-Fas antibody-mediated apoptosis was observed in this system.
Hirofumi Akari, Tomoharu Fukumori and Akio Adachi : Cell-dependent requirement of human immunodeficiency virus type 1 gp41 cytoplasmic tail for Env incorporation into virions., Journal of Virology, Vol.74, No.10, 4891-4893, 2000.
(Summary)
Growth kinetics in lymphocytic H9 and M8166 cells of two mutants of human immunodeficiency virus type 1 (HIV-1) with deleted gp41 cytoplasmic tails were examined. While the mutant viruses designated CTdel-44 and CTdel-144 were able to grow in M8166 cells, they were unable to grow in H9 cells. Transfection and single-round infectivity assays demonstrated that they are defective in the early phase of viral replication in H9 cells. Analysis of the mutant virions revealed drastically reduced incorporation of Env gp120 (compared with the incorporation of wild-type virions) in H9 cells but normal incorporation in M8166 cells. These results indicate that the HIV-1 cytoplasmic tail of gp41 determines virus infectivity in a cell-dependent manner by affecting incorporation of Env into virions and suggest the involvement of a host cell factor(s) in the Env incorporation.
(Keyword)
Cell Line / HIV Envelope Protein gp120 / HIV Envelope Protein gp41 / HIV-1 / Humans / Mutation / Virion / Virus Assembly / Virus Replication
Hajime A Koyama, Tsutomu Arakawa and Akio Adachi : Characterization of apoptosis induced by sorbitol: a unique system for the detection of antiapoptotic activities of viruses, Microbes and Infection, Vol.2, No.6, 599-606, 2000.
(Summary)
The treatment of HEp-2 cells with sorbitol induced massive apoptosis rapidly. This method for inducing apoptosis is very useful to detect antiapoptotic activity of viruses as well as viral genes. Commitment to death occurred immediately upon incubation with sorbitol, even in the presence of pancaspase inhibitor, Z-VAD-FMK. Apoptosis is also induced by other polyhydric alcohols with more than four hydroxyl groups, but not induced by glycerol or ethylene glycol. Sorbitol treatment on ice did not induce apoptosis either. These results suggest that this induction of apoptosis does not result simply from high osmotic pressure but probably by the interaction of solutes through their physical nature (such as hydrophobicity) with the plasma membrane of the cells.
Hirofumi Akari, Stefan Arold, Tomoharu Fukumori, Toshiyuki Okazaki, Klaus Strebel and Akio Adachi : Nef-Induced Major Histocompatibility Complex Class I Down-Regulation Is Functionally Dissociated from Its Virion Incorporation, Enhancement of Viral Infectivity, and CD4 Down-Regulation, Journal of Virology, Vol.74, No.6, 2907-2912, 2000.
(Summary)
The N-terminal alpha-helix domain of the human immunodeficiency virus type 1 (HIV-1) Nef protein plays important roles in enhancement of viral infectivity, virion incorporation of Nef, and the down-regulation of major histocompatibility complex class I (MHC-I) expression on cell surfaces. In this study, we demonstrated that Met 20 in the alpha-helix domain was indispensable for the ability of Nef to modulate MHC-I expression but not for other events. We also showed that Met 20 was unnecessary for the down-regulation of CD4. These findings indicate that the region governing MHC-I down-regulation is proximate in the alpha-helix domain but is dissociated functionally from that determining enhancement of viral infectivity, virion incorporation of Nef, and CD4 down-regulation.
(Keyword)
Amino Acid Sequence / Antigens, CD4 / Base Sequence / Cells, Cultured / Down-Regulation / Gene Products, nef / Genes, MHC Class I / HIV-1 / HeLa Cells / Humans / Leukocytes, Mononuclear / Methionine / Molecular Sequence Data / Virion / nef Gene Products, Human Immunodeficiency Virus
Akiko Yoshida, J Shimomoto, KB Koh, Mikako Fujita and Akio Adachi : The H9/M8166 tropism of various HIV-1 mutants is determined by distinct cellular factors, International Journal of Molecular Medicine, Vol.5, No.3, 291-294, 2000.
(Summary)
We have previously shown that a number of human immunodeficiency virus type 1 (HIV-1) mutants generated in vitro display a replication-defect in a cell-dependent manner. Of the mutants of this category, those of gag, vif, and env mutants do not grow at all in lymphocytic H9 (non-permissive) cells, but quite well in M8166 (permissive) cells. To determine whether the cell-dependent growth of the mutants is functionally related to each other, a number of double mutants were constructed, and monitored for their replication and biochemical property in various cells. The results obtained have indicated that there are multiple cellular factors responsible for the growth phenotype. Together with our previous findings on the host cell-dependent mutants of HIV-1, it is concluded that number of distinct cellular factors are involved in the various steps in HIV-1 replication cycle.
Hirofumi Akari, Stefan Arold, Tomoharu Fukumori, Toshiyuki Okazaki, Klaus Strebel and Akio Adachi : Nef-induced major histocompatibility complex class 1 down-regulation is functionally dissociated from its virion incorporation, enhancement of virul infectibity, and CD4 down-regulation., Journal of Virology, Vol.74, No.6, 2907-2912, 2000.
116.
Yoshitaka Isaka, Akihiko Sato, Shigeru Miki, Shinobu Kawauchi, Hitoshi Sakaida, Toshiyuki Hori, Takashi Uchiyama, Akio Adachi, Masanori Hayami, Tamio Fujiwara and Osamu Yoshie : Small Amino Acid Changes in the V3 Loop of Human Immunodeficiency Virus Type 2 Determines the Coreceptor Usage for CXCR4 and CCR5, Virology, Vol.264, No.1, 237-243, 1999.
(Summary)
HIV-2 GH-1 is a molecular clone derived from an AIDS patient from Ghana. In contrast to the prototypic molecular clone ROD, GH-1 exhibits a narrow range of target cell specificity. By an infectious assay using HeLa-CD4 cells stably transfected with an HIV-1 LTR-beta-galactosidase reporter gene and transiently expressing various cloned chemokine receptors, we have examined the coreceptor usage of GH-1. In contrast to ROD, which uses principally CXCR4, GH-1 was found to use mainly if not exclusively CCR5 but not CXCR4. The distinct coreceptor usage of these two molecular clones allowed us to further map the region of gp120 that is important for the coreceptor specificity. By constructing a series of chimeric viruses between GH-1 and ROD, we have demonstrated that the C-terminal half of the V3 loop region of gp120 determines the differential coreceptor usage between GH-1 and ROD, and only a few amino acid differences in this region appear to be able to shift the specificity between CCR5 and CXCR4. Notably, the shift in the coreceptor usage from CCR5 to CXCR4 is associated with an increase in the net positive charge in the V3 region.
Hirofumi Akari, Tsuneo Uchiyama, Tomoharu Fukumori, Shinya Iida, Hajime A Koyama and Akio Adachi : Pseudotyping human immunodeficiency virus type 1 by vesicular stomatitis virus G protein does not reduce the cell-dependent requirement of Vif for optimal infectivity: functional difference between Vif and Nef, The Journal of General Virology, Vol.80, No.11, 2945-2949, 1999.
(Summary)
The functions of Vif and Nef in human immunodeficiency virus type 1 (HIV-1) infection have some similarities: Vif- and Nef-dependent enhancement of HIV-1 replication is cell type-specific, and defective mutations in these genes result in restricted proviral DNA synthesis in infected cells. It has recently been shown that pseudotyping HIV-1 by the envelope glycoprotein of vesicular stomatitis virus (VSV-G) targets HIV-1 entry to an endocytic pathway and suppresses the requirement of Nef for virus infectivity. In this study, we examined whether VSV-G pseudotyping suppresses the requirement of Vif for HIV-1 infectivity. It was found that pseudotyping HIV-1 by VSV-G did not compensate for the Vif function. Together with the findings that Vif does not influence virus binding/entry and virion incorporation of Env, it is concluded that Vif enhances HIV-1 infectivity at the post-entry step(s) independently of the Env function by a different mechanism to that of Nef.
Hajime A Koyama, Tsutomu Arakawa and Akio Adachi : Comparison of an antiviral activity of recombinant consensus interferon with recombinant interferon-α-2b, Microbes and Infection, Vol.1, No.13, 1073-1077, 1999.
(Summary)
To avoid possible uncertainty in comparing biological activities of interferon samples from different sources where interferon concentrations were determined independently, we prepared chromatographically pure preparations of consensus interferon and interferon-alpha-2b (one of the two commercially available recombinant alpha interferons). We revealed that consensus interferon has a stronger antiviral activity than interferon-alpha-2b, although the effects of these two recombinant interferons on the cellular macromolecule synthesis are at similar levels.
Hirofumi Akari, Tomoharu Fukumori, Shinya Iida and Akio Adachi : Induction of Apoptosis in Herpesvirus saimiri-Immortalized T Lymphocytes by Blocking Interaction of CD28 with CD80/CD86, Biochemical and Biophysical Research Communications, Vol.263, No.2, 352-356, 1999.
120.
Akio Adachi, Michio Tamaki, Reika Shimano, Ritsuko Inubushi, Takehiro Naito, Kazuko Yoshida, Yoko Oshima, Meiko Kawamura and Hajime A Koyama : Cell-dependent replication potentials of HIV-1 gag mutants, Microbes and Infection, Vol.1, No.9, 671-676, 1999.
(Summary)
An infectious molecular clone of human immunodeficiency virus type 1 (HIV-1), designated pNLaiKH, which is tropic for both lymphocytic and monocytic cells, was constructed. To study the early function of HIV-1 Gag proteins in two types of cells, the mutations known to give host cell-dependent early defects were introduced into pNLaiKH, and the replication potentials and defective replication sites in the cells of the resultant mutants were monitored. All mutants grew in some lymphocytic cells, but not at all in monocytic cells. A nucleocapsid mutant was found to be defective at an early replication phase in all the cell lines to various extent, as expected. In contrast, a matrix mutant and a capsid mutant displayed a replication defect in a producer-cell-dependent manner. These results demonstrated that complex interactions of cell factors and Gag proteins are involved in an early process of HIV-1 replication.
So Hata, Hajime A Koyama, Hiroshi Shiota, Akio Adachi, Fumi Goshima and Yukihiro Nishiyama : Antiapoptotic activity of herpes simplex virus type 2: the role of US3 protein kinase gene, Microbes and Infection, Vol.1, No.8, 601-607, 1999.
(Summary)
In order to determine the ability of herpes simplex virus type 2 (HSV-2) to suppress apoptosis, we examined the effect of HSV-2 infection on apoptosis induced in HEp-2 cells by treatment with 1 M sorbitol. Although a wild-type strain of HSV-2 induced apoptosis in a significant fraction of the infected cells, HSV-2 could suppress sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that HSV-2, like HSV-1, has an antiapoptosis gene. Characterization of the cells infected with a US3-deletion mutant of HSV-2 revealed the necessity of a US3 gene in the antiapoptotic activity of this virus.
(Keyword)
herpes simplex virus / apoptosis / antiapoptosis / protein kinase
Reika Shimano, Ritsuko Inubushi, Yoko Oshima and Akio Adachi : Inhibition of HIV/SIV Replication by Dominant Negative Gag Mutants, Virus Genes, Vol.18, No.3, 197-201, 1999.
(Summary)
There are several major strategies against HIV/AIDS. Of these, the gene therapy is a novel, challenging, and promising one. The target genes, which have been extensively studied for the potential gene therapy of HIV/AIDS, include those of cellular and viral origins. Especially, trans-dominant negative Tat, Rev, Env, Pol, and Gag mutants of HIV have currently attracted considerable attention. In this brief review, we summarize the nature of the HIV/SIV mutants of this category and discuss their future use for gene therapy with special reference to the dominant negative Gag mutants of HIV-1.
Hirofumi Akari, Ki-Hoan Nam, Kazuyasu Mori, Isao Otani, Hiroaki Shibata, Akio Adachi, Keiji Terao and Yasuhiro Yoshikawa : Effects of SIVmac Infection on Peripheral Blood CD4+CD8+T Lymphocytes in Cynomolgus Macaques, Clinical Immunology, Vol.91, No.3, 321-329, 1999.
(Summary)
We have previously reported that CD4+CD8+ double-positive (DP) T cells with a resting memory phenotype exist in a substantial proportion of peripheral blood lymphocytes of adult cynomolgus macaques. In this study, we examined the effects of simian immunodeficiency virus of macaque (SIVmac) infection on DP T cells. In vitro, SIVmac239 nef-open (239) and its nef-deletion mutant replicated well in both CD4+CD8- and DP T cells. However, when the macaques were infected with 239, DP, but not CD4+CD8-, T cells were transiently increased in parallel with cell activation and viral replication, followed by depletion within 1 month postinfection. Interestingly, the nef gene was required for depletion but not for the increase and activation of DP T cells. These data suggest that the pathogenic SIV infection may downmodulate production and/or blood circulation of DP T cells by a Nef function-related mechanism(s) different from that for the depletion of CD4+CD8- T cells.
Reika Shimano, Ritsuko Inubushi and Akio Adachi : Host cell-dependent replication of HIV-1 gag MA, CA, and NC mutants are independent of the functions of Vif and Vpu, International Journal of Molecular Medicine, Vol.3, No.1, 91-94, 1999.
(Summary)
We have previously shown that some gag gene mutants of human immunodeficiency virus type 1 (HIV-1) display a replication-defect in a cell-dependent manner. We and others have also demonstrated that the requirement of vif and vpu genes for HIV-1 replication is cell-dependent. To determine whether the cell-dependent growth of the HIV-1 gag mutants is related to the functions of Vif and Vpu, double mutants of gag-vif and gag-vpu were constructed, and monitored for their replication in various cell lines. The results obtained showed that the mutations in gag do not affect the cell-dependent functions of Vif and Vpu.
(Keyword)
Capsid / Capsid Proteins / Cell Line / Gene Products, gag / Gene Products, vif / HIV Antigens / HIV Core Protein p24 / HIV-1 / Human Immunodeficiency Virus Proteins / Humans / Mutation / Recombinant Fusion Proteins / Tumor Cells, Cultured / Viral Proteins / Viral Regulatory and Accessory Proteins / Virus Replication / gag Gene Products, Human Immunodeficiency Virus / vif Gene Products, Human Immunodeficiency Virus
Kenzo Tokunaga, Kazuyoshi Ikuta, Akio Adachi, Michiyuki Matsuda, Takeshi Kurata and Asato Kojima : The Cellular Kinase Binding Motifs (PxxP and RR) in Human Immunodeficiency Virus Type 1 Nef Protein Are Dispensable for Producer-Cell-Dependent Enhancement of Viral Entry, Virology, Vol.257, No.2, 285-289, 1999.
(Summary)
We have recently demonstrated that human immunodeficiency virus type 1 (HIV-1) Nef is required for enhancing viral infectivity by increasing the efficiency of viral entry in a producer-cell-dependent manner, suggesting the possible involvement of a cellular factor(s) in the enhancement of viral entry. Moreover, it has been reported that a proline-rich (PxxP) motif and an Arg-Arg (RR) motif in HIV-1 Nef bind to the SH3 domain of the Src-family tyrosine kinase Hck and to a serine/threonine kinase, respectively. To address whether these cellular kinase binding motifs, PxxP and RR, could be involved in virus producer-cell-dependent enhancement of viral entry, we constructed two nef mutant proviral clones in which these motifs were mutated. The results show that the HIV-1 Nef PxxP motif, which significantly influenced viral infectivity, and the RR motif, which modestly affected viral infectivity, were both dispensable for enhanced viral entry, thus suggesting that another interaction of Nef with a cellular factor(s) is involved in the efficiency of viral entry.
Ritsuko Inubushi and Akio Adachi : Cell-dependent function of HIV-1 Vif for virus replication, International Journal of Molecular Medicine, Vol.3, No.5, 473-476, 1999.
(Summary)
It has been well established that the Vif protein of human immunodeficiency virus type 1 (HIV-1) acts late in the viral life cycle and increases the infectivity of the progeny virions in a producer cell-dependent manner. The virions produced in the absence of Vif in non-permissive cells (Delta Vif) are defective for a step(s) before and/or during reverse transcription. In this review, the functional and structural analyses of these virions including our new data are summarized.
Shinya Iida, Tomoharu Fukumori, Yoko Oshima, Hirofumi Akari, Hajime A Koyama and Akio Adachi : Compatibility of Vpu-like Activity in the Four Groups of Primate Immunodeficiency Viruses, Virus Genes, Vol.18, No.2, 183-187, 1999.
128.
Koji Sakai, Miyuki Horiuchi, Shinya Iida, Tomoharu Fukumori, Hirofumi Akari and Akio Adachi : Mutational Analysis of Human Immunodeficiency Virus Type 1 Vif Gene, Virus Genes, Vol.18, No.2, 179-181, 1999.
(Summary)
Mutations were introduced into scattered regions of the HIV-1 vif gene. The twelve in-frame mutants generated were evaluated for the replication potentials in cells by transfection and infection experiments. All the mutants produced a normal level of progeny virions upon transfection, indicating the absence of the late function of HIV-1 Vif protein. The infectivity of virions obtained was monitored in H9 cells, which are non-permissive for HIV-1 without the Vif function. Most of the mutations in various parts of the vif gene, including those in the three conserved regions among HIV/SIV, abrogated the infectivity of the virus. In contrast, the cysteine residue at position 133, which was reported to be critical for viral infectivity, was found not to be essential. In addition, the C-terminal eight amino acid residues (185-192) in the Vif protein could be deleted with no effects on viral growth potential.
Akio Adachi and Yoko Oshima : Cell-dependent functional roles of HIV-1 Nef for virus replication, International Journal of Molecular Medicine, Vol.3, No.4, 427-430, 1999.
130.
Akio Adachi, Yoko Oshima and Hajime A Koyama : Activation of HIV-1 enhancer sequence by vaccinia virus, International Journal of Molecular Medicine, Vol.3, No.3, 311-313, 1999.
(Summary)
To investigate whether vaccinia virus (VV) can augment gene expression of human immunodeficiency virus type 1 (HIV-1), co-transfection experiments were carried out in which recombinant plasmids containing various portions of the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into cultured cells. A high level of enhancement in CAT activity directed by the HIV-1 LTRs containing the enhancer sequence was observed in cells infected with VV, as in the cells infected with type 1 herpes simplex virus (HSV-1). The sequence responsible for this augmentation of CAT activity was different from that recognized by HIV-1 Tat. These data clearly demonstrated that VV transactivates HIV-1 LTR through a mechanism distinct from that of activation by HIV-1 Tat.
(Keyword)
Animals / Cell Line / Gene Expression Regulation, Viral / Gene Products, tat / Genes, Reporter / Genes, Viral / Genetic Vectors / HIV Enhancer / HIV-1 / Herpesvirus 1, Human / Humans / Kinetics / Sequence Deletion / Transcriptional Activation / Transfection / Vaccines, Synthetic / Vaccinia virus / tat Gene Products, Human Immunodeficiency Virus
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 10028058
Tomoharu Fukumori, Susumu Kagawa, Shinya Iida, Yoko Oshima, Hirofumi Akari, Hajime A Koyama and Akio Adachi : Rev-dependent expression of three species of HIV-1 mRNAs, International Journal of Molecular Medicine, Vol.3, No.3, 297-302, 1999.
(Summary)
The expression of structural and accessory genes of human immunodeficiency virus type 1 (HIV-1) except for nef requires a viral regulatory protein Rev. Rev-dependency of the expression of structural (gag, pol and env), regulatory (tat and rev), and accessory genes (vif, vpr, vpu and nef) has been investigated by various systems, and it has been demonstrated that unspliced (encodes gag and pol) and singly-spliced (env-vpu, vif and vpr) viral mRNAs are differentially dependent on the function of Rev. In this review, the function of HIV-1 Rev in relation to these findings is discussed.
Shinya Iida, Tomoharu Fukumori, Yoko Oshima, Hajime A Koyama and Akio Adachi : Growth characteristics of T-cell tropic HIV-1 vpu gene mutants in human peripheral blood mononuclear cells, The Journal of Medical Investigation : JMI, Vol.46, No.1-2, 43-47, 1999.
(Summary)
A mutant designated NL-E65, which lacks the expression of entire vpu gene, was constructed from T-cell tropic wild-type (wt) human immunodeficiency virus type 1 (HIV-1) clone and monitored for its replication property in human cells, along with a mutant NL-Ss which expresses a C-terminal truncated Vpu. The mutant NL-Ss could grow in two cell lines and in all peripheral blood mononuclear cell (PBMC) preparations to some extent, with kinetics similar to those of wt virus. Likewise, the mutant NL-E65 exhibited a replication property typical to the vpu mutant in the two cell lines and in all PBMC cultures, growing at a low level. Along with the results previously reported, these data indicate that HIV-1 Vpu is dispensable for virus replication in any of the types of cells so far tested.
(Tokushima University Institutional Repository: 83261, PubMed: 10408156)
133.
Akio Adachi, Shinya Iida, Tomoharu Fukumori, Michio Tamaki, Ritsuko Inubushi, Reika Shimano, Yoko Oshima, Hirofumi Akari and Hajime A Koyama : Exchangeability of accessory Vif and Vpu proteins between various HIV/SIVs, International Journal of Molecular Medicine, Vol.3, No.2, 193-197, 1999.
134.
Setsuko Ishikawa, Seiichi Hashida, Kazuya Hashinaka, Akio Adachi, Shinichi Oka and Eiji Ishikawa : Rapid formation of the immune complexes on solid phase in the immune complex transfer enzyme immunoassay for HIV-1 p24 antigen and antibody IgGs to HIV-1, Journal of Clinical Laboratory Analysis, Vol.12, No.4, 227-237, 1998.
(Summary)
In order to perform the immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17, reverse transcriptase and gp41 antigens as rapidly as possible, methods for rapid formation of the immune complexes on solid phase are described. HIV-1 p24 antigen was reacted with monoclonal anti-p24 Fab'-beta-D-galactosidase conjugate at a high concentration and subsequently with polystyrene beads coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl bovine serum albumin-affinity-purified anti-p24 Fab' conjugate. Antibody IgGs to HIV-1 were reacted with polystyrene beads coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-HIV-1 antigen conjugates and subsequently with HIV-1 antigen-beta-D-galactosidase conjugates. The periods of time used for the formation of the immune complexes comprising the three components on the polystyrene beads (15-30 min) were much shorter than those used in the previous immune complex transfer enzyme immunoassays (90-300 min), and the sensitivities of the present and previous immune complex transfer enzyme immunoassays were similar. The detection limit of the HIV-1 p24 antigen by the present and previous methods were similar (3 to 10 zmol/assay).
(Keyword)
antibody / human immunodeficiency virus type 1 / enzyme immunoassay / d-galactosidase
Setsuko Ishikawa, Seiichi Hashida, Kazuya Hashinaka, Akio Adachi, Shinichi Oka and Eiji Ishikawa : Ultrasensitive and rapid enzyme immunoassay (thin aqueous layer immune complex transfer enzyme immunoassay) for antibody IgG to HIV-1 p17 antigen, Journal of Clinical Laboratory Analysis, Vol.12, No.3, 179-189, 1998.
(Summary)
The immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 p17 antigen was performed in two different ways (the present immunoassays I and II) within shorter periods of time than previously reported. In the present (simultaneous) immunoassay I, antibody IgG to HIV-1 p17 antigen in 10 microL of serum samples was incubated simultaneously with 2,4-dinitrophenyl-maltose binding protein-recombinant p17(rp17) fusion protein and rp17-beta-D-galactosidase conjugate in a total volume of 22 microL for 10 min to form the immune complex comprising the three components. The reaction mixture was incubated with a polystyrene bead of 6.35 mm in diameter coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 5 min in a styrol test tube (13.3 x 54 mm and 2.1 g) to trap the immune complex. After washing, the polystyrene bead was incubated with 30 microL of epsilonN-2,4-dinitrophenyl-L-lysine solution in a polystyrene tube (12 x 75 mm) coated with affinity-purified (antihuman IgG gamma-chain) IgG for 10 min to transfer the immune complex. In the present (sequential) immunoassay 11, a polystyrene bead of 6.35 mm in diameter coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-maltose binding protein-rp17 fusion protein was incubated in a styrol test tube (13.3 x 54 mm and 2.1 g) sequentially with antibody IgG to HIV-1 p17 antigen in 10 microL of serum samples in a total volume of 16 microL for 5 min and subsequently with rp17-beta-D-galactosidase conjugate in a volume of 10 microL for 5 and 10 min. The immune complex formed on the polystyrene bead was transferred to a polystyrene tube coated with affinity-purified (antihuman IgG gamma-chain) IgG for 5 and 10 min in the same way as in the present immunoassay I. During the incubations, the styrol test tubes containing the polystyrene beads and reaction mixtures were shaken, and the polystyrene test tubes were rotated with shaking, so that the polystyrene beads were rotated randomly, and small drops (16 to 30 microL) of the reaction mixtures evenly contacted all parts of the solid phase surfaces during the incubations, though only small parts of the solid phase surfaces were contacted at one time. The intent was to continuously mix thin aqueous layers of the reaction mixtures covering the solid phase surfaces with the rest of the reaction mixtures. (Therefore, these immunoassays were called thin aqueous layer immunoassays.) beta-D-Galactosidase activity bound to the polystyrene tubes was assayed by fluorometry for 30 and 60 min. The present immunoassays I and II, in which only 15 to 25 min were used for the immunoreactions, were as sensitive if not more so than the previous immune complex transfer enzyme immunoassay requiring 150 min for the immunoreactions. In these earlier immunoreactions, the immune complex comprising the three components formed by 30 min incubation was trapped onto two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 60 min, and was then transferred to two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (antihuman IgG y-chain) IgG for 60 min in a total volume of 150 microL. Furthermore, the present (sequential) immunoassay 11 (and probably I) could become approximately 10 times more sensitive by assaying bound beta-D-galactosidase activity for a longer period of time (10 h), since beta-D-galactosidase activity, bound nonspecifically in the presence of serum samples from HIV-1 seronegative subjects, was considerably low.
(Keyword)
antibody / human immunodeficiency virus type 1 / p17 / enzyme immunoassay
Hajime A Koyama, Hirofumi Akari, Akio Adachi, Fumi Goshima and Yukihiro Nishiyama : Induction of apoptosis in HEp-2 cells by infection with herpes simplex virus type 2, Archives of Virology, Vol.143, No.12, 2435-2441, 1998.
(Summary)
Although herpes simplex virus type 1 (HSV-1) does not induce apoptosis in infected HEp-2 cells, herpes simplex virus type 2 (HSV-2) did induce apoptosis in a small but significant fraction of the same cells. Apoptosis was not observed in Vero or HeLa cells infected with HSV-2. In addition, HSV-2 infection in the presence of cycloheximide induced extensive apoptosis of HEp-2 or HeLa cells.
(Keyword)
Animals / apoptosis / Cell Line / Cercopithecus aethiops / Cycloheximide / Cytopathogenic Effect, Viral / DNA Fragmentation / HeLa Cells / Herpesvirus 1, Human / Herpesvirus 2, Human / Humans / Protein Synthesis Inhibitors / Vero Cells
Reika Shimano, Ritsuko Inubushi, Michio Tamaki, Hirofumi Akari, Hajime A Koyama and Akio Adachi : The non-env tropism of HIV/SIV, International Journal of Molecular Medicine, Vol.2, No.5, 541-544, 1998.
(Summary)
The tropism of human and simian immunodeficiency viruses (HIV and SIV) determined by sequences other than env has been studied. The restriction of HIV-1 replication in monkey cells was demonstrated to be regulated by viral non-env sequence. Likewise, the gag-pol region of SIVagm (virus of the African green monkey) genome was found to be responsible for growth restriction in human cells of the virus. No viral DNA synthesis was detected in cells nonpermissive for the viruses. In addition, a number of HIV-1 gag gene mutants, which have an early defect in viral replication cycle and direct no viral DNA synthesis in some cells, exhibited a phenotype of host range mutant. Taken together, it can be concluded that the viral tropism associated with the uncoating/ reverse transcription process does exist in HIV/SIV replication. Furthermore, many of the accessory gene mutants of HIV/SIV exhibit host cell-dependent replication property. In this review, we summarize these examples of non-env tropism of HIV/SIV.
Ritsuko Inubushi, Michio Tamaki, Reika Shimano, Hajime A Koyama, hirofumi Akari and Akio Adachi : Functional roles of HIV accessory proteins for viral replication, International Journal of Molecular Medicine, Vol.2, No.4, 429-433, 1998.
(Summary)
Numerous lentiviruses, including human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) of causative agents of human AIDS as representative, have been recently isolated from various species of primates. The fundamental and most prominent feature of the viruses is the presence of a number of accessory genes in their genomes. Extensive biological and biochemical studies have demonstrated that the accessory gene products are not essential for viral replication at least in certain types of cells. Quite surprisingly, some of these accessory proteins are absolutely non-essential in any types of cells so far examined. In this brief review, our systematic genetic studies on the importance of the accessory proteins of HIV-1 and HIV-2 for viral replication are described and discussed.
Kenzo Tokunaga, Asato Kojima, Takeshi Kurata, Kazuyoshi Ikuta, Hirofumi Akari, Hajime A Koyama, Meiko Kawamura, Ritsuko Inubushi, Reika Shimano and Akio Adachi : Enhancement of human immunodeficiency virus type 1 infectivity by Nef is producer cell-dependent, The Journal of General Virology, Vol.79, No.10, 2447-2453, 1998.
(Summary)
The growth kinetics of wild-type and nef mutant viruses of human immunodeficiency virus type 1 were comparatively analysed in several human CD4+ cell lines. Delayed replication of nef mutant virus was observed in all cell lines examined. To determine the stage in the virus replication cycle that is affected by Nef, a single-round replication assay was performed. Initially, the expression of marker genes in transfected cells was examined in order to study the role of Nef in the late phase of infection. The results obtained indicated that Nef is dispensable during the transcription to virion production stage. Next, the effect of Nef on the early phase was investigated with a single-round infection. It was demonstrated that Nef is required in the early phase of the virus replication cycle, from virion adsorption to integration. Finally, the infectivity of virus stocks prepared from four cell lines was determined. The relative infectivity of the nef mutant from the four cell lines differed. Taken together, we conclude that Nef acts via modulation of viral particles to enhance virus infectivity in a cell-dependent manner.
Kenzo Tokunaga, Asato Kojima, Takeshi Kurata, Kazuyoshi Ikuta, Ritsuko Inubushi, Reika Shimano, Meiko Kawamura, Hirofumi Akari, Hajime A Koyama and Akio Adachi : Producer Cell-Dependent Requirement of the Nef Protein for Efficient Entry of HIV-1 into Cells, Biochemical and Biophysical Research Communications, Vol.250, No.3, 565-568, 1998.
(Summary)
A proviral nef gene mutant of human immunodeficiency virus type 1 (HIV-1) was evaluated for its defective early replication step. Virus stocks were prepared from six CD4-positive and -negative cell lines transfected with wild-type (wt) or the nef mutant clone and inoculated into two target CD4-positive cell lines to monitor the efficiency of viral entry process. The nef mutant virions produced in one cell line exhibited a severe defect in the entry process, although those produced in the other five cell lines were only slightly less efficient than the wt virions at entering into cells. These results have demonstrated that the HIV-1 Nef is critical for efficient viral entry in a producer cell-dependent manner.
(Keyword)
CD4-Positive T-Lymphocytes / Cell Line / Gene Products, nef / HIV Infections / HIV-1 / Humans / Virus Replication / nef Gene Products, Human Immunodeficiency Virus
Ritsuko Inubushi, Reika Shimano, Yoko Oshima, Kazuko Yoshida, Meiko Kawamura and Akio Adachi : Suppression of HIV replication by dominant negative mutants of HIV-1, International Journal of Molecular Medicine, Vol.2, No.3, 325-330, 1998.
(Summary)
Various gag mutants of human immunodeficiency virus type 1 (HIV-1) generated in vitro were evaluated for their potentials to suppress the replication of wild-type (wt) virus. A single-round of wt virus replication in the presence of various mutant proteins was quantitatively monitored by transfection and infection experiments. Out of 38 mutants examined, 15 were demonstrated to interfere with the replication of wt HIV-1 at early and/or late viral replication phase. Some of these mutants were also effective against the replication of wt HIV-2. In this review, we focus on the mutants, which are able to act against a wide variety of HIV, and are very useful for future gene therapy against AIDS.
(Keyword)
HIV-1 / Humans / Mutation / Virus Replication
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 9855705
Tomoharu Fukumori, Hirofumi Akari, Shinya Iida, So Hata, Susumu Kagawa, Yoko Aida, Hajime A Koyama and Akio Adachi : The HIV-1 Vpr displays strong anti-apoptotic activity, FEBS Letters, Vol.432, No.1-2, 17-20, 1998.
(Summary)
Mutations in the human immunodeficiency virus type 1 (HIV-1) vpr gene only slightly reduce the replication rate of the virus. To study the role of HIV-1 Vpr in biological effects on cells, HEp-2 cells, which express HIV-1 Vpr constitutively but at a low level, were established. While control HEp-2 cells underwent apoptosis when incubated with sorbitol, the morphological and biochemical apoptotic changes were inefficiently induced in the HIV-1 Vpr-expressing cells by the same treatment. These results clearly indicate that HIV-1 Vpr has anti-apoptotic activity, and raise the possibility that Vpr acts as a weak activator of virus replication through anti-apoptosis.
Meiko Kawamura, Reika Shimano, Ritsuko Inubushi, Hirofumi Akari and Akio Adachi : Early Function of HIV-1 Gag Proteins Is Cell-Dependent, Biochemical and Biophysical Research Communications, Vol.248, No.3, 899-903, 1998.
(Summary)
Various gag gene mutants of human immunodeficiency virus type 1 (HIV-1) were monitored for their replication potentials and defective replication sites in various CD4-positive T-cell lines. Some matrix, capsid, and nucleocapsid mutants displayed a replication defect in a cell-dependent manner. The single-round replication assays demonstrated that these mutants were defective at an early infection phase also in a cell-dependent way. These results indicated that interaction of a cell factor(s) and Gag proteins is involved in an early process of HIV-1 replication.
Reika Shimano, Ritsuko Inubushi, Tomoharu Fukumori, Michio Tamaki, Yoko Oshima, Meiko Kawamura and Akio Adachi : Suppression of HIV-2 Replication by HIV-1gagMutants, Biochemical and Biophysical Research Communications, Vol.248, No.2, 418-421, 1998.
(Summary)
Gag gene mutants of human immunodeficiency virus type 1 (HIV-1) were analyzed for their potentials of inhibiting the replication of wild-type (wt) HIV-2, the second AIDS virus, in a single-round of viral replication. Of twenty-two HIV-1 gag mutants examined, seven were found to efficiently interfere with the replication of wt HIV-2. Some mutants, which can suppress the replication of wt HIV-1, did not show this inhibitory effect. These mutants were defective at the late phase of viral replication. A mutant designated NL-C1a was demonstrated to be very effective against the replication of HIV-1 and HIV-2 in monocytic cells as well as in lymphocytic cells.
Ritsuko Inubushi, Reika Shimano, Yoko Oshima and Akio Adachi : The Potential of Various HIV-1 Mutants to Inhibit the Replication of Wild-Type Virus, Biochemical and Biophysical Research Communications, Vol.247, No.2, 349-352, 1998.
(Summary)
We have previously demonstrated that many of gag mutants of human immunodeficiency virus type 1 (HIV-1) inhibited the replication of wild-type (wt) HIV-1. In this study, various HIV-1 mutants were systematically analyzed with respect to their ability to suppress the replication of wt HIV-1. Sixteen mutants of all eight HIV-1 genes other than gag were evaluated for their inhibitory effects. Only an env mutant designated NL-Hi efficiently interfered with the replication of wt HIV-1 in a single round of infection. The NL-Hi did not affect the late replication processes of wt virus, including transcription, translation, and assembly/release. Virions produced in the presence of the mutant Env were defective for the viral entry process in the early phase of HIV-1 replication cycle.
Hirofumi Akari, Kazuyasu Mori, Isao Otani, Keiji Terao, Fumiko Ono, Akio Adachi and Yasuhiro Yoshikawa : Induction of MHC-II DR expression on circulating CD8+ lymphocytes in macaques infected with SIVmac239 nef-open but not with its nef-deletion mutant, AIDS Research and Human Retroviruses, Vol.14, No.7, 619-625, 1998.
(Summary)
We examined the expression kinetics of activation antigens CD38 and MHC-IIDR (DR) on circulating CD8+ lymphocytes in rhesus macaques infected with pathogenic simian immunodeficiency virus strain SIVmac239 nef-open (239) or its nonpathogenic nef-deletion mutant (delta nef). In the longitudinal study, we found for the first time the induction of DR expression on CD8+ lymphocytes in 239-infected macaques. The induction of DR was in parallel with an increasing viral load and a decreasing CD4+ lymphocyte level. In the macaques with the high viral load and low CD4 level, a considerable proportion of the DR+CD8+ subpopulation was CD69+, indicating an activated state. On the other hand, no significant increase in the DR+CD8+ subpopulation level was observed in delta nef-infected macaques. These data indicate that the evaluation of activation markers such as DR and/or CD69 on circulating CD8+ cells may be valuable as a surrogate marker in the SIV-macaque model.
Hajime A Koyama, Tsutomu Arakawa and Akio Adachi : Acceleration of virus-induced apoptosis by tumor necrosis factor, FEBS Letters, Vol.426, No.2, 179-182, 1998.
(Summary)
The multiplication of vesicular stomatitis virus in HeLa cells was inhibited by treating the cells with tumor necrosis factor (TNF). Comparison of the kinetics of virus multiplication and that of virus-induced apoptosis in the TNF-treated cells revealed that the antiviral effect of TNF is accompanied by a rapid induction of apoptosis in the cells upon infection, suggesting that TNF can inhibit virus multiplication by accelerating an apoptotic response in the infected cells.
Hirofumi Akari, Hideo Yagita, Tadashi Nishida, Kenji Nakamaru, Yasuhiro Yoshikawa and Akio Adachi : Selective induction of β7 integrin expression on lymphocytes undergoing apoptosis in lymphoid tissues, Biochemical and Biophysical Research Communications, Vol.244, No.2, 578-582, 1998.
(Summary)
It has been previously shown that the beta 7 chain of integrin forms heterodimers with the alpha 4 or alpha E chain, which plays essential roles in lymphocyte homing to mucosal lymphoid tissues. The aim of this study was to re-evaluate the possible role of the beta 7 integrin other than lymphocyte homing. We prepared spleen and lymph node lymphocytes from biopsied specimens from macaque monkeys and examined for the reactivity with a monoclonal antibody specific for the beta 7 chain. As a result, a minor population of the lymphocytes with a smaller size, which were in the early stage of apoptosis, was found to express a higher level of the beta 7 integrin than a majority of the lymphocytes with a normal size. Interestingly, the apoptotic lymphocytes expressed neither alpha 4 nor alpha E chains, suggesting that the beta 7 chain on these cells may be associated with an undefined alpha chain. These findings indicate that in the lymphoid tissues the shrunken lymphocytes undergoing apoptosis selectively express a unique beta 7 integrin.
Hirofumi Akari, Fumiko Ono, Ippei Sakakibara, Hikaru Takahashi, Yuichi Murayama, Akio Hiyaoka, Keiji Terao, Isao Otani, Ryozaburo Mukai, Akio Adachi and Yasuhiro Yoshikawa : Simian T cell leukemia virus type I-induced malignant adult T cell leukemia-like disease in a naturally infected African green monkey: implication of CD8+ T cell leukemia, AIDS Research and Human Retroviruses, Vol.14, No.4, 367-371, 1998.
(Summary)
Spontaneous T cell leukemia was found in an African green monkey (Cercopithecus aethiops, AGM) naturally infected with simian T cell leukemia virus type I (STLV-I). The hematological features and the evidence for monoclonal integration of provirus DNA in the leukemic cells revealed that the leukemia was an ATL-like disease. The expression of surface markers on the leukemic cells indicated that they were defined as an activated CD8+ T cell subset. Together with the finding that seven in vitro spontaneously STLV-I-transformed cell lines were CD4-CD8+, it is likely that CD8+ T cells are transformed by STLV-I in AGMs, in contrast with human ATL. Finally, we assessed characteristics of the CD8 chains on these transformed cells. The result indicated that the leukemic cells expressed only the alpha chains but not the beta chains. However, in the case of in vitro-transformed cell lines the expression pattern of the CD8 chains varied in individual monkeys. Thus, STLV-I may preferentially transform CD8+ (both alphaalpha+ and alphabeta+) T cells in AGMs.
Meiko Kawamura, Reika Shimano, Takashi Ogasawara, Ritsuko Inubushi, Kazushi Amano, Hirofumi Akari and Akio Adachi : Mapping the genetic determinants of human immunodeficiency virus type 2 for cell tropism and replication efficiency, Archives of Virology, Vol.143, No.3, 513-521, 1998.
(Summary)
Two distinct infectious molecular clones of human immunodeficiency type 2 (HIV-2) were analyzed for their biological properties in six cell lines. Fourteen chimeric and ten mutant viruses were constructed from these two viral genomes to localize the genetic determinants responsible for the phenotypes. Growth property of the viruses in the cell lines, together with the biochemical data, showed that a major determinant for the viral tropism resides in the env gene. In addition, in some cell lines, the accessory genes vif and nef affected the efficiency of virus replication. Thus, like HIV-1, mutations in the auxiliary and env genes of HIV-2 contributed much to the differences in virological characteristics.
(Keyword)
Base Sequence / Cell Line, Transformed / Chromosome Mapping / DNA, Viral / Gene Products, tat / HIV-2 / HL-60 Cells / Humans / Molecular Sequence Data / Recombination, Genetic / Tumor Cells, Cultured / Virus Replication / tat Gene Products, Human Immunodeficiency Virus
Hirofumi Akari, Yoshihiko Yamamoto, Hiroko Hiwada, Yasuyoshi Saito, Noriyasu Takayanagi, Hajime A Koyama and Akio Adachi : Suppression of HIV-1 replication in peripheral blood mononuclear cells by fasudil, The Journal of Medical Investigation : JMI, Vol.44, No.3-4, 211-214, 1998.
(Summary)
Fasudil is a potent inhibitor for various protein kinases such as myosin light chain kinase and protein kinase C. It has been used as a drug for improvement of intracranial vasospasm and following ischaemic diseases. In this report, we demonstrate that fasudil suppressed the replication of human immunodeficiency virus type 1 (HIV-1) in mitogen-activated peripheral blood mononuclear cells. Our finding shows that fasudil may be useful as a new and distinct chemotherapeutic agent against HIV-1 infection.
(Tokushima University Institutional Repository: 110669, PubMed: 9597811, Elsevier: Scopus)
152.
Reika Shimano, Shinya Iida, Tomoharu Fukumori, Yoshihiko Yamamoto, Meiko Kawamura, Rika A. Furuta and Akio Adachi : Inhibition of HIV Replication by Capsid Mutant C6b, Biochemical and Biophysical Research Communications, Vol.242, No.2, 313-316, 1998.
(Summary)
A Gag capsid mutant of human immunodeficiency virus type 1 (HIV-1) designated C6b was biologically and biochemically characterized with respect to its ability to suppress the replication of wild-type (wt) HIV. The C6b efficiently interfered with the replication of wt HIV-1 in the cleavage of Gag precursor, and also in the early replication process before or during viral DNA synthesis after viral penetration. The C6b Gag appeared to be unable to form chimeric multimers with HIV-2 Gag and failed to inhibit the replication of wt HIV-2.
Reika Shimano, Ritsuko Inubushi, Kazushi Amano, Takashi Ogasawara, Hirofumi Akari, Hajime A Koyama, Meiko Kawamura and Akio Adachi : Complete Inhibition of SIVmac Replication by its Capsid Mutants, Virus Genes, Vol.17, No.1, 43-48, 1998.
154.
Reika Shimano, Ritsuko Inubushi, Kazushi Amano, Takashi Ogasawara and Akio Adachi : Gag Pol Region Determines the Tropism of SIVagm for Human Cells, Virus Genes, Vol.16, No.2, 137-139, 1998.
(Summary)
Simian immunodeficiency virus isolated from African green monkeys (SIVagm) does not grow in many of human cell lines such as CEM x 174, H9, and MT-4, but could replicate in some human cell lines. Sequence of SIVagm responsible for its narrow host range was determined by making and monitoring growth potential of chimeric clones between SIVagm and human immunodeficiency virus type 1 (HIV-1). The results obtained indicated that the gag-pol region determines the observed narrow host range. By monitoring virus DNA synthesis and progeny virion production, the defect(s) of SIVagm in the replication in the restricted cells was demonstrated to be located at early phase.
Hirofumi AKARI, Keiji TERAO, Ki-Hoan NAM, Akio Adachi and Yasuhiro YOSHIKAWA : Comparative Analysis of Human and Macaque Monkey CD4: Differences in Formaldehyde Lability and Conformation, Experimental Animals, Vol.47, No.1, 23-27, 1998.
(Summary)
Here we characterized macaque monkey CD4 by flow cytometry. The results showed that relatively lower fluorescence intensity was observed depending on the monoclonal antibodies (mAbs) used for staining; Leu-3a exhibited four-fold lower intensity than Nu-Th/i, and that formaldehyde fixation dramatically reduced fluorescence intensity of macaque CD4+ cells stained with Leu-3a but not of human cells. Nu-Th/i is therefore preferable for the analysis of macaque CD4. Pretreatment of either mAb inhibited the other mAb binding to human CD4. On the contrary, Nu-Th/i inhibited Leu-3a binding but Leu-3a poorly blocked Nu-Th/i binding to the macaque CD4. These results indicate that Leu-3a and Nu-Th/i epitopes are conserved in macaque CD4 but Leu-3a epitope is conformationally cryptic and/or fragile, resulting in the lower affinity. Amino acid sequence alignment of CD4 domain 1 shows that the substitutions outside the linear Leu-3a epitope may determine these characteristics of macaque CD4.
Meiko Kawamura, Reika Shimano, Ritsuko Inubushi, Kazushi Amano, Takashi Ogasawara, Hirofumi Akari and Akio Adachi : Functional Domain Mapping of HIV-1 Gag Proteins, Biochemical and Biophysical Research Communications, Vol.241, No.2, 317-320, 1997.
(Summary)
A series of human immunodeficiency virus type 1 (HIV-1) proviral gag gene mutants carrying bacterial CAT gene were constructed and monitored for the expression of reverse transcriptase and CAT in a highly sensitive single-round replication assay system to determine the defective replication phase in lymphocytic cells. All the mutants displayed no abnormality in the process of transcription and translation at late replication stage. In contrast, some matrix, capsid, and p6 mutants were defective at final phase, that is, assembly and virion release. Most of the mutants including nucleocapsid mutants, which showed normal phenotype at late stage, were defective at early replication phase. From the functional domain map thus obtained, it is evident that HIV-1 Gag proteins are required for both early and late replication phases.
Hajime A Koyama and Akio Adachi : Induction of apoptosis by herpes simplex virus type 1, The Journal of General Virology, Vol.78, 2909-2912, 1997.
(Summary)
Although herpes simplex virus type 1 (HSV-1) does not induce apoptosis in infected HEp-2 cells, in the presence of cycloheximide infection induced apoptosis with characteristic morphological changes as well as endonucleosomal DNA cleavage. The induction of apoptosis without de novo protein synthesis suggests that a structural protein of the HSV-1 virion is responsible for the observed apoptosis.
(Keyword)
apoptosis / Cell Line / Herpes Simplex / Herpesvirus 1, Human / Humans / Viral Structural Proteins
Rika A. Furuta, Reika Shimano, Takashi Ogasawara, Ritsuko Inubushi, Kazushi Amano, Hirofumi Akari, Masakazu Hatanaka, Meiko Kawamura and Akio Adachi : HIV-1 capsid mutants inhibit the replication of wild-type virus at both early and late infection phases, FEBS Letters, Vol.415, No.2, 231-234, 1997.
(Summary)
In-frame mutations were introduced into various portions of the human immunodeficiency virus type 1 (HIV-1) gag gene, and potentials of the mutants to suppress the replication of wild-type HIV-1 were monitored. In contrast to results obtained with matrix and nucleocapsid mutants, almost all capsid mutants blocked HIV-1 replication completely in single-round replication assays. A capsid mutant designated C6b was demonstrated to be one of the most efficient inhibitors for HIV-1 reported to date, and to be effective at both early and late viral replication phases. T-cells, which are engineered to express the C6b Gag in response to HIV-1 infection, were perfectly resistant to HIV-1.
Meiko Kawamura, Reika Shimano, Ritsuko Inubushi, Kazushi Amano, Takashi Ogasawara, Hirofumi Akari and Akio Adachi : Cleavage of Gag precursor is required for early replication phase of HIV-1, FEBS Letters, Vol.415, No.2, 227-230, 1997.
(Summary)
A mutant of human immunodeficiency virus type 1 (HIV-1), which is deficient for Gag precursor cleavage and noninfectious, was characterized with respect to its defective step in the viral replication phase. Upon transfection, the mutant produced a normal level of progeny virions as monitored by electron microscopy and RNA hybridization. Single-round replication assay demonstrated, in contrast, that the mutant was defective at the early phase of the replication cycle. Furthermore, no viral DNA was detected in the cells infected with the mutant. Taken together, it is concluded that maturation of Gag precursor protein of HIV-1 is required for an early event(s) before or during a coupled process of uncoating/reverse transcription.
Tatsuhiko Igarashi, Yasushi Ami, Hiroshi Yamamoto, Riri Shibata, Takeo Kuwata, Ryozaburo Mukai, Katsuaki Shinohara, Toshihiko Komatsu, Akio Adachi and Masanori Hayami : Protection of monkeys vaccinated with vpr- and/or nef-defective simian immunodeficiency virus strain mac/human immunodeficiency virus type 1 chimeric viruses: a potential candidate live-attenuated human AIDS vaccine, The Journal of General Virology, Vol.78, No.Pt5, 985-989, 1997.
(Summary)
Two simian immunodeficiency virus strain mac (SIVmac)/human immunodeficiency virus type 1(HIV-1) chimeric viruses (SHIVs), designated NM-3 and NM-3n, with env derived from HIV-1 and defective vpr (plus defective nef for NM-3), were inoculated into seven macaques. These macaques were transiently or persistently infected and most of them produced long-lasting neutralizing antibodies and Env-specific killer T cells to HIV-1 with no AIDS-like symptoms. When they were challenged with another SHIV with intact vpr and nef (designated NM-3rN), all were protected as judged by virus recovery, DNA detection by PCR and antibody responses. Anti-HIV-1 Env-specific killer T cells were considered to have played a major role in this protection, but a non-specific defence mechanism as well as specific immunity also appeared to be involved. Thus, these two non-pathogenic SHIVs induced long-lasting protective immunities in macaques, suggesting the possibility of gene-defective SHIVs as attenuated live vaccines for human use.
Saburo Sone, Yoshihiko Yamamoto, Akio Adachi, Jin Asano, Shinya Iida and Yasuyoshi Saito : Functional Analysis of vif Genes Derived from Various Primate Immunodeficiency Viruses, Virus Genes, Vol.14, No.3, 195-200, 1997.
(Summary)
Replication property in cells of human and simian immunodeficiency viruses (HIVs and SIVs) lacking intact vif gene was evaluated. Of 10 vif mutants constructed in vitro of the major four HIV/SIV groups, only those derived from HIV-1 and HIV-2/SIVmac displayed replication defect. The cell lines non-permissive for the vif mutants of HIV-1 and SIVmac were found to be different. To determine whether Vif is exchangeable between HIV-1 and SIVmac, chimeric virus clones with respect to the vi gene were constructed and virus replication in the cells non-permissive for the vif mutant viruses was monitored. Productive infection in these cells of chimeric viruses clearly indicated that Vif is functionally exchangeable, and that Vifs of different virus origin act through a similar mechanism.
Kyoko Higo, Yoshinao Kubo, Yasumasa Iwatani, Takeshi Ono, Michiyuki Maeda, Horoshi Hiai, Tohru Masuda, Kagemasa Kuribayashi, Fengmin Zhang, Ta Yar Lamin, Akio Adachi and Akinori Ishimoto : Susceptibility of nude mice carrying the Fv-4 gene to Friend murine leukemia virus infection, Journal of Virology, Vol.71, No.1, 750-754, 1997.
(Summary)
Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). Despite complete resistance to ecotropic MuLV infection in mice carrying the Fv-4 gene, it is known that cells carrying the resistance gene in tissue culture do not always show resistance as extensive as that in vivo (H. Yoshikura and T. Odaka, JNCI 61:461-463, 1978). To investigate the immunological effect on resistance in vivo, we introduced the Fv-4 gene into BALB/c nude mice (Fv-4-/- nude[nu/nu]) by mating them with Fv-4 congenic BALB/c mice (Fv-4r/r nude+/+) and examined the susceptibility of the F2 progeny to F-MuLV. All BALB/c nude mice without the Fv-4 gene (Fv-4-/- nude[nu/nu]) were permissive to F-MuLV and developed erythroleukemia within 2 weeks after virus inoculation. The BALB/c nude mice with the Fv-4 gene (Fv-4r/r nude[nu/nu]) did not develop leukemia, and no or little virus was detected in the spleen 7 weeks after virus inoculation. The resistance to F-MuLV was dominant in (Fv-4 congenic BALB/c x BALB/c nude) F1 mice with the Fv-4r/- nude(nu/+) genotype as strictly as in (Fv-4 congenic BALB/c x BALB/c) F1 mice with the Fv-4r/- nude+/+ genotype. However, almost all BALB/c nude mice with the Fv-4r/- nude(nu/nu) genotype developed the disease within 7 weeks, and the virus was detected in all of their spleens even in the mice without leukemia. These results show that the resistance caused by the Fv-4 gene is recessive in nude mice and dominant in BALB/c mice. Some immunological effects, perhaps cell-mediated immunity, may play important roles in the resistance to F-MuLV infection in vivo in addition to the dosage effect of the Fv-4 product.
Kaori Otake, Yoichi Fujii, Masato Tashiro, Akio Adachi and Junzoh Kitoh : Epitope mapping of murine monoclonal antibodies against human immunodeficiency virus type 1 Nef, Experimental Animals, Vol.46, No.1, 53-58, 1997.
(Summary)
It has shown that the human immunodeficiency virus type 1 (HIV-1) Nef protein has the high antigenicity in HIV-1 seropostive individuals. We newly obtained seven monoclonal antibodies (mAbs). To identify the antigenic determinants of HIV-1 Nef protein against murine, epitope mapping of the mAbs was performed by enzyme-linked immunosorbent assay (ELISA) by using several recombinant truncated Nef fusion proteins, that were expressed in Esherichia coli, and synthetic peptides. The results showed that mAbs A6, A7, F2, F3, F4, F8 and E5 recognized epitopes on Nef protein located at amino acid residues 18-26, 28-45, 115-137, 128-137, 115-126, 128-137, and 170-181, respectively.
Kenzo Tokunaga, A Ishimoto, K Ikuta and Akio Adachi : Growth ability of auxiliary gene mutants of human immunodeficiency virus types 1 and 2 in unstimulated peripheral blood mononuclear cells, Archives of Virology, Vol.142, No.1, 177-181, 1997.
(Summary)
Mutational studies on the Vif, Vpr, Vpu, Vpx, and Nef genes of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) were performed to evaluate their biological functions in natural target cells. For this purpose, replication properties of mutant viruses derived from HIV-1 NL strain and HIV-2 GH strain in unstimulated peripheral blood mononuclear cells were determined. Vif- viruses of both HIV-1 and HIV-2 did not grow at all in these cells. Similarly, no replication of HIV-2 Vpx- mutant was detected. In contrast, both of Vpr- and Nef- viruses of HIV-1 and HIV-2, and Vpu- virus of HIV-1 grew quite well in the cells. These results show, together with the data previously reported, that only Vif and Vpx are essential for HIV replication in primary blood cell cultures.
Yoichi Fujii, Kaori Otake, Masato Tashiro and Akio Adachi : In vitro cytocidal effects of human immunodeficiency virus type 1 Nef on unprimed human CD4+ T cells without MHC restriction, The Journal of General Virology, Vol.77, No.12, 2943-2951, 1996.
(Summary)
We have previously shown that the C-terminal region of human immunodeficiency virus type 1 (HIV-1) Nef antigen present on the outer surface of virus-infected cells has an affinity for uninfected T cells and that the Nef protein is responsible for T cell death. To exclude completely the possibility of MHC restriction of this cytotoxic activity, the in vitro cytotoxic potential of HIV-1 Nef against various CD4+ T cell lines as well as naive T lymphocytes was investigated using a baculovirus expression system. Insect cells expressing myristoylated Nef on their cell surface were shown to kill a proportion of CD4+ T cells within 8 h. However, N-terminal truncated and unmyristoylated Nef proteins were not present on the outer surface of insect cell membranes and failed to show any killing activity. Monoclonal antibodies against the C-terminal region of Nef inhibited cytolysis. Thus, we conclude that specific Nef-mediated cytolysis is induced by contact with unprimed CD4+ T lymphocytes without MHC restriction.
Yoichi Fujii, Kaori Otake, Yoshikazu Fujita, Naoki Yamamoto, Yoshiyuki Nagai, Masato Tashiro and Akio Adachi : Clustered localization of oligomeric Nef protein of human immunodeficiency virus type 1 on the cell surface, FEBS Letters, Vol.395, No.2-3, 257-261, 1996.
(Summary)
We studied human immunodeficiency virus type 1 (HIV-1) Nef protein biochemically and histologically. HIV-1 Nef, derived from baculosystem and from cells infected with HIV-1, formed homomeric monomers, dimers, trimers, and further polymers. These oligomers were non-covalently associated. In cells infected with HIV-1, Nef molecules were clustered at the cell surface as well as cytoplasm. Our previous results have indicated that the Nef on the surface of cells infected with HIV-1 is cytotoxic against uninfected CD4+ T cells. Thus, it is very likely that the HIV-1-mediated cytotoxic reaction is due, at least in part, to the clustered localization of oligomeric Nef on the cell surface.
Yoichi Fujii, Kaori Otake, Masato Tashiro and Akio Adachi : Human immunodeficiency virus type 1 Nef protein on the cell surface is cytocidal for human CD4+ T cells, FEBS Letters, Vol.393, No.1, 105-108, 1996.
(Summary)
We have previously shown that the carboxyl-terminal region of human immunodeficiency virus type 1 (HIV-1) Nef antigen present on the outer surface of virus-infected cells has affinity for uninfected T cells. Here, the in vitro cytotoxic potential of HIV-1 Nef on the T cell surface against CD4+ T cells was investigated in detail. Human T cells expressing Nef on the cell surface by transfection with non-infectious mutant HIV-1 proviruses were demonstrated to kill CD4+ T cells efficiently. Furthermore, it was shown that the carboxyl-terminal portion of Nef was cytotoxic for CD4+ T cells and that monoclonal antibody against the carboxyl-terminal region of Nef inhibited Nef induced-cytolysis. Thus, we concluded that Nef protein on CD4+ T cells may play an important role in the specific loss of CD4+ T lymphocytes during HIV-1 infection.
Yoichi Fujii, Kaori Otake, Masato Tashiro and Akio Adachi : Soluble Nef antigen of HIV-1 is cytotoxic for human CD4+ T cells, FEBS Letters, Vol.393, No.1, 93-96, 1996.
(Summary)
We have previously shown that Nef-gene 10 fusion protein induces marked growth arrest of human primary CD4+ T cells. Here, in vitro cytostatic and cytotoxic activities of human immunodeficiency virus type 1 (HIV-1) Nef against CD4+ T cells were extensively investigated. Growth of human CD4+ cells was inhibited significantly just by the addition of purified full-length Nef to cultures. When Nef was cross-linked by anti-Nef antibodies, it became very cytocidal for CD4+ T cells. A high percentage of sera from HIV-1-infected individuals contained soluble Nef. Thus, soluble Nef in vivo may play an important role in immunodysfunction of CD4+ T lymphocytes in HIV-1 infection.
(Keyword)
CD4-Positive T-Lymphocytes / cell division / Cell Line / Cytotoxicity Tests, Immunologic / Gene Products, nef / HIV Antigens / HIV Infections / HIV-1 / Humans / nef Gene Products, Human Immunodeficiency Virus
Yoshinao Kubo, Kazuhiro Kakimi, Kyoko Higo, Hirohiko Kobayashi, Takeshi Ono, Yuko Iwama, Kagemasa Kuribayashi, Hiroshi Hiai, Akio Adachi and Akinori Ishimoto : Possible origin of murine AIDS (MAIDS) virus: conversion of an endogenous retroviral p12gag sequence to a MAIDS-inducing sequence by frameshift mutations, Journal of Virology, Vol.70, No.9, 6405-6409, 1996.
(Summary)
The murine AIDS (MAIDS) virus has a unique sequence in its p12gag region, which is responsible for MAIDS development. A transcript hybridizing with this sequence is expressed in normal C57BL/6 mice. The transcript, designated Edv, has been previously cloned and sequenced (Y. Kubo, Y. Nakagawa, K. Kakimi, H. Matsui, K. Higo, L. Wang, H. Kobayashi, T. Hirama, and A. Ishimoto, J. Gen. Virol. 75:881-888, 1994). Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic replication-defective MAIDS virus has a 16-bp deletion and a 1-bp insertion in the 5' and 3' regions of the p12gag sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the defective MAIDS virus p12gag region is not homologous to that of the helper virus and the Edv transcript because of the frameshift. To determine whether the amino acid sequence resulting from the frameshift is critical for MAIDS development, we constructed chimeric viruses that contained the p12gag regions of the helper virus and the Edv transcript, respectively, with and without the same frame as the defective MAIDS virus by the artificial frameshift mutations. The mutant viruses with the frameshift mutations induced MAIDS in inoculated mice, but the viruses without the mutations did not. These results suggested that the MAIDS virus was generated by frameshift mutations in the p12gag region of Edv or a related sequence.
Tatsuhiko Igarashi, Takeo Kuwata, Jun Takehisa, Kentaro Ibuki, Riri Shibata, Ryozaburo Mukai, Toshihiko Komatsu, Akio Adachi, Eiji Ido and Masanori Hayami : Genomic and biological alteration of a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac chimera with HIV-1 Env recovered from a long-term carrier monkey, The Journal of General Virology, Vol.77, No.8, 1649-1658, 1996.
(Summary)
A macaque monkey infected with NM-3, a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac (SIVmac) chimeric virus with env, rev, tat and vpu derived from HIV-1 and LTR, gag, pol, vif and vpx derived from SIVmac, became a long-term carrier (more than 2.8 years). This monkey produced neutralizing antibodies to the original NM-3 as well as to the parental HIV-1. The virus recovered at 116 weeks replicated more rapidly and productively in macaque peripheral blood mononuclear cells than the original virus. The recovered virus was not neutralized either by antibodies raised early in the monkey or by a neutralizing monoclonal antibody that recognizes the V3 loop of HIV-1 Env, whereas both the early antibodies and the monoclonal antibody neutralized the original NM-3. Analysis of the virus genomic population revealed a few common mutations in the V3 region that caused amino acid changes. These data are consistent with the hypothesis that the virus escaped from the early antibodies and that the observed mutations contributed to this, as with HIV-1-infected humans. The observed mutations could equally well be the result of adaptation to simian cells. These results suggest that the HIV-1-SIVmac chimeric virus will be useful for investigating genetic variation of HIV-1 env and alteration of biological properties in vivo in relation to the host immune response.
(Keyword)
Amino Acid Sequence / Animals / Base Sequence / Blood Transfusion / Carrier State / Cell Line / DNA, Viral / Genes, env / Genetic Variation / HIV Antibodies / HIV Infections / HIV-1 / Humans / Kinetics / Macaca fascicularis / Male / Molecular Sequence Data / Mutation / Neutralization Tests / Reassortant Viruses / Simian Acquired Immunodeficiency Syndrome / Simian Immunodeficiency Virus / Virus Latency
Akihiko Sato, Jun Yoshimoto, Yoshitaka Isaka, Shigeru Miki, Akemi Suyama, Akio Adachi, Masanori Hayami, Tamio Fujiwara and Osamu Yoshie : Evidence for direct association of Vpr and matrix protein p17 within the HIV-1 virion, Virology, Vol.220, No.1, 208-212, 1996.
(Summary)
Vpr is one of the auxiliary proteins of HIV-1 and is selectively incorporated into the virion by a process involving the C-terminal p6 portion of the Gag precursor Pr55. Vpr and the matrix protein p17 are the components of the viral preintegration complex and appear to play important roles in the nuclear transport of proviral DNA in nondividing cells. In the present study, we have demonstrated by coimmunoprecipitation experiments that Vpr associates with matrix protein p17 but not with capsid protein p24 within the HIV-1 virion. Experiments employing the yeast two-hybrid GAL4 assay for protein-protein interactions also demonstrated a direct association between Vpr and the C-terminal region of matrix protein p17. Association of Vpr and the matrix protein p17 within the mature virion is consistent with their collaborative role in the nuclear transportation of the viral preintegration complex in nondividing cells such as macrophages.
(Keyword)
Animals / Base Sequence / Cell Line / Cricetinae / DNA, Viral / Gene Products, gag / Gene Products, vpr / Guinea Pigs / HIV Antigens / HIV-1 / Humans / Molecular Sequence Data / Precipitin Tests / Tumor Cells, Cultured / Viral Proteins / gag Gene Products, Human Immunodeficiency Virus / vpr Gene Products, Human Immunodeficiency Virus
T. Miura, Riri Shibata, Akio Adachi, T. Kuwata, J. Chen, M Jin, E. Ido and Masanori Hayami : Genetic complementation between replication-defective mutants of HIV-1 and SIVagm, Archives of Virology, Vol.141, No.1, 31-41, 1996.
(Summary)
To investigate the functional complementation of essential genes for virus growth between HIV-1 and SIVagm derived from African green monkeys, we co-transfected replication-defective molecular clones containing mutations in gag, pol, env, tat or rev, and monitored transient complementation by reverse transcriptase assay (RT), cytopathic effect (CPE) and immunofluorescence assay (IFA). The following results were obtained: 1) No complementation was observed in combinations of the gag and pol mutants. 2) The rev mutant of HIV-1 was minimally complemented by other SIVagm mutants, although the rev mutant of SIVagm was significantly complemented by other HIV-1 mutants. 3) Among all combinations tested, the env mutant of HIV-1 was the most effectively complemented by SIVagm mutants. 4) CPE was mostly absent in combinations of the env mutant of SIVagm and the gag, pol, or tat mutants of HIV-1, although there were significant positive results in RT and IFA assays. These findings provided basic information about the functional compatibility of pathogenic HIV-1 and nonpathogenic SIVagm which will be useful for generating chimeras of these two viruses.
(Keyword)
Animals / Cell Line / Cercopithecus aethiops / Genes, env / Genes, gag / Genes, pol / Genes, rev / Genes, tat / Genetic Complementation Test / HIV Reverse Transcriptase / HIV-1 / Humans / Mutation / RNA-Directed DNA Polymerase / Simian Immunodeficiency Virus / Tumor Cells, Cultured / Virus Replication
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 8629949
Junichi Sakuragi, Sayuri Sakuragi, Shigeharu Ueda and Akio Adachi : Functional analysis of simian immunodeficiency virus SIVagm long terminal repeat, Virus Genes, Vol.12, No.1, 21-25, 1996.
(Summary)
We have previously shown that long terminal repeats (LTRs) derived from various isolates of SIVAGM share a unique functional property. In the absence of viral Tat, all SIVAGM LTRs act as much more efficient promoters than any of the other LTRs derived from representative primate immunodeficiency viruses. In the presence of Tat, however, SIVAGM LTRs are activated relatively inefficiently. To map the elements that confer these features on the SIVAGM LTR, a number of deletion mutants were constructed, and their promoter activities were determined using a bacterial CAT gene as a marker. The results obtained indicated that various elements located in the U3 region may contribute to the high basal promoter activity and that no negative elements are present in the region. The Tat-responsive sequence TAR was localized to the R region as observed for the other LTRs. A mutant carrying a single nucleotide deletion in this region completely lost responsiveness to Tat protein.
(Keyword)
Base Sequence / DNA, Viral / Gene Products, tat / Molecular Sequence Data / Nucleic Acid Conformation / RNA, Viral / Repetitive Sequences, Nucleic Acid / Simian Immunodeficiency Virus
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 8879117
S. Sakuragi, J. Sakuragi and Akio Adachi : Both SU and TM Env proteins are responsible for monkey cell tropism of simian immunodeficiency virus SIVmac, Archives of Virology, Vol.140, No.12, 2255-2260, 1995.
(Summary)
While simian immunodeficiency virus (SIV) derived from an infectious molecular clone pMA239 is tropic and pathogenic for monkeys, the virus derived from another infectious clone pMA142 does not replicate in monkey cells. To determine genetic sequences responsible for this tropism, a series of recombinant clones were constructed from pMA142 and pMA239. The determinant in pMA239 was mapped within regions encompassing the env gene. Viruses, which carry the 239 env gene encoding surface and/or transmembrane proteins, were tropic for monkey cells.
Akihiko Sato, Yoshitaka Isaka, Makoto Kodama, Jun Yoshimoto, Shinobu Kawauchi, Takeo Kuwata, Akio Adachi, Masanori Hayami, Osamu Yoshie and Tamio Fujiwara : Targeting of chrolamphenicol acetyltransferase to human immunodeficiency virus particles via Vpr and Vpx, Microbiology and Immunology, Vol.39, No.12, 1015-1019, 1995.
(Summary)
Vpr and Vpx are the auxiliary proteins of human immunodeficiency viruses (HIVs) selectively incorporated into mature viral particles. We showed that the bacterial chloramphenicol acetyltransferase (CAT) fused to the N-terminus of HIV-1 Vpr, HIV-2 Vpr, or HIV-2 Vpx was incorporated into mature virions in a type-selective manner. By using chimeric proteins between HIV-1 Vpr and HIV-2 Vpx, we found that the N-terminal side of these proteins was mainly important for type-selective virion incorporation. The C-terminal arginine-rich region of HIV-1 Vpr was also found to transport CAT fusion proteins into virions but without any type selectivity. Furthermore, the corresponding regions of HIV-2 Vpr and HIV-2 Vpx had no such activity. This region of HIV-1 Vpr may interact nonspecifically with viral genomic RNA. Collectively, Vpr and Vpx may provide a means to introduce foreign proteins and other molecules into HIV virions for therapeutic purposes.
Riri Shibata, Hiroyuki Sakai, Meiko Kawamura, Kenzo Tokunaga and Akio Adachi : Early replication block of human immunodeficiency virus type 1 in monkey cells, The Journal of General Virology, Vol.76, No.11, 2723-2730, 1995.
(Summary)
The genetic and functional basis of the replication-defective nature of human immunodeficiency virus type 1 (HIV-1) in monkey cells was studied. By the generation and characterization of chimeras between HIV-1 and simian immunodeficiency virus, the sequence encompassing the 3' half of the long terminal repeat, gag and pol genes of HIV-1 was found to be responsible for the growth restriction. Early and late phases of HIV-1 replication in monkey cells were analysed in detail using several assay systems: transfection/coculture, transcomplementation between various proviral clones carrying the CAT gene and effector clones and evaluation of transcription and reverse transcription. All the data were consistent with the notion that HIV-1 replication is blocked at a very early stage(s) such as uncoating and/or reverse transcription in monkey cells.
(Keyword)
Animals / Base Sequence / Cells, Cultured / DNA Primers / DNA, Viral / Defective Viruses / Genes, gag / Genes, pol / HIV Long Terminal Repeat / HIV-1 / Haplorhini / Humans / Leukocytes, Mononuclear / Molecular Sequence Data / Recombination, Genetic / Simian Immunodeficiency Virus / Transcription, Genetic / Virus Replication
Hiroyuki Sakai, Kenzo Tokunaga, Meiko Kawamura and Akio Adachi : Function of human immunodeficiency virus type 1 Vpu protein in various cell types, The Journal of General Virology, Vol.76, No.11, 2717-2722, 1995.
(Summary)
We evaluated the function of human immunodeficiency virus type 1 vpu gene in various cell lines. We established a highly sensitive system consisting of chloramphenicol acetyltransferase and reverse transcriptase assays and used it to monitor the effects of mutation of the vpu gene. In some cell lines, Vpu protein was not required at the early phase of viral replication but was important for efficient virion production. In these cells, the Vpu protein functioned effectively irrespective of the presence of intact env gene products. Likewise, CD4 gene expression had no effects on Vpu function. In the other cell lines tested, Vpu protein was not important for virion release, and the vpu mutant clone generated a normal level of progeny virions upon transfection.
(Keyword)
3T3 Cells / Animals / CD4-Positive T-Lymphocytes / Cell Line / Genes, vpu / HIV-1 / HeLa Cells / Human Immunodeficiency Virus Proteins / Humans / Mice / Mutation / Viral Regulatory and Accessory Proteins / Virus Replication
Jun-ichi Sakuragi, Hiroyuki Sakai, Meiko Kawamura, Kenzo Tokunaga, Shigeharu Ueda and Akio Adachi : Generation and characterization of a host cell-dependent gag gene mutant of human immunodeficiency virus type 1, Virology, Vol.212, No.1, 251-254, 1995.
(Summary)
An in-frame gag gene mutant of human immunodeficiency virus type 1, which carries two amino acid substitutions in the center of the p24 coding region, was constructed in vitro, and its replication properties in several cell lines were examined. In CD4-negative SW480 cells transfected with the mutant clone, synthesis and processing of viral gag, pol, and env proteins occurred normally, and viral particles were produced. Virions derived from the transfection displayed a severe replication defect when inoculated into some CD4-positive cell lines (H9 and Molt4 clone 8), but in other lines (A3.01 and M8166), the mutant virus grew fairly well. The mutant was demonstrated to be defective at an early infection phase (from adsorption to integration) in Molt4 clone 8 cells but was normal in A3.01 cells. These results indicated that the Gag-p24 protein of human immunodeficiency virus type 1 plays an important role at the early infection phase in a cell-dependent manner.
(Keyword)
Amino Acid Sequence / Base Sequence / CD4-Positive T-Lymphocytes / Cell Line / Defective Viruses / Genes, gag / HIV Core Protein p24 / HIV-1 / Humans / Molecular Sequence Data / Virus Replication
Hiroyuki Sakai, Rika A. Furuta, Kenzo Tokunaga, Meiko Kawamura, Masakazu Hatanaka and Akio Adachi : Rev-dependency of expression of human immunodeficiency virus type 1 gag and env genes, FEBS Letters, Vol.365, No.2-3, 141-145, 1995.
(Summary)
Structural gene expression of human immunodeficiency virus type 1 (HIV-1) requires a viral regulatory protein, Rev transactivator. We investigated Rev-dependency of HIV-1 gene expression by various reporter systems. Expression of unspliced and single-spliced viral mRNAs was demonstrated to be differentially dependent on the Rev function. This difference of Rev-dependency was found not to be determined by cis-elements in gag, pol, and env coding sequences reported so far, and was lost when the reporter constructs containing minimum elements for Rev-responsiveness such as splice signals and rev responsive element were used for experiments. These findings indicated that the fundamental structure of HIV-1 mRNA was critical for the differential regulation of gene expression by Rev transactivator.
Jun Katahira, Toshimasa Ishizaki, Hiroyuki Sakai, Akio Adachi, Kazuhiko Yamamoto and Hisatoshi Shida : Effects of translation initiation factor eIF-5A on the functioning of human T-cell leukemia virus type I Rex and human immunodeficiency virus Rev inhibited trans dominantly by a Rex mutant deficient in RNA binding, Journal of Virology, Vol.69, No.5, 3125-3133, 1995.
(Summary)
The viral transactivator proteins Rex and Rev are necessary for the expression of structural proteins of human T-cell leukemia virus type I and human immunodeficiency virus type 1, respectively. Although the interaction of Rex/Rev with a cellular cofactor(s) has been thought to be required for Rex/Rev action, there is no suitable system to search for the cofactor(s) in mammalian cells. We found that a Rex mutant, TAgRex, which contains a simian virus 40 nuclear localization signal in place of the N-terminal 19 amino acids of Rex, could dominantly inhibit wild-type Rex/Rev functions. The inhibition did not require either Rev response element/Rex response element binding or the oligomerization ability of the mutant, but it did require a region around amino acid 90 of the Rex protein, suggesting that TAgRex sequestered the cellular cofactor. Complementation with the eukaryotic translation initiation factor 5A (eIF-5A) in this system could restore the impaired Rex function. These results indicate that eIF-5A is the cofactor indispensable for Rex function. Additionally, by using a two-hybrid system, the homo-oligomer formation of Rex was found to be mediated by the region around amino acid 90 in addition to Tyr-64 and Trp-65 of Rex protein. Thus, eIF-5A may play a part in the formation of the Rex homo-oligomer.
(Keyword)
Animals / Base Sequence / Cell Line / DNA Primers / DNA, Viral / Gene Products, rev / Gene Products, rex / Genes, Dominant / HIV-1 / Human T-lymphotropic virus 1 / Humans / Molecular Sequence Data / Mutation / Peptide Initiation Factors / RNA, Viral / RNA-Binding Proteins / rev Gene Products, Human Immunodeficiency Virus
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 7707541
Akio Adachi, Hiroyuki Sakai, Kenzo Tokunaga and Meiko Kawamura : Functional analysis of human spuma retrovirus genome, Virus Genes, Vol.11, No.1, 15-20, 1995.
(Summary)
Human spuma retrovirus (HSRV) belongs to retroviruses that possess a complex genome organization. HSRV carries at least three extra genes in the region between env and the 3' long terminal repeat, which are not found in simple retroviruses. Via alternative splicing, these HSRV genes can encode several proteins. To genetically study the requirements of these viral proteins for viral replication in tissue cultures, a number of mutant viruses were constructed from an infectious molecular clone of HSRV. All mutants grew normally in the cell lines tested, except for those lacking transcriptional transactivator activity. By reporter-based transient assay systems, no Rev/Rex equivalent was detected in the HSRV proteins.
Rika A Furuta, Hiroyuki Sakai, Meiko Kawamura, Kenzo Tokunaga, Masakazu Hatanaka and Akio Adachi : Functionality of chimeric Rev proteins of HIV/SIV, Virus Genes, Vol.11, No.1, 11-14, 1995.
(Summary)
Studies on functional compatibility of various Rev proteins derived from all known human and simian immunodeficiency virus subgroups have shown that this essential gene product is not always exchangeable among the viruses. In an attempt to map the region of Rev proteins responsible for the observed nonreciprocal complementation, hybrid genomic Rev expression vectors were constructed by exchanging the first and second exons of rev genes, and were examined for their abilities to activate reporter clones by transfection. With one exception, the second coding exon of rev gene determined the functional specificity of Rev proteins.
Meiko Kawamura, Hiroyuki Sakai and Akio Adachi : Human imunodeficiency virus Vpx is required for the early phase of replication in peripheral blood mononuclear cells, Microbiology and Immunology, Vol.38, No.11, 871-878, 1994.
(Summary)
Functional importance of Vpx protein of human immunodeficiency virus type 2 was evaluated in various types of cells. In 8 lymphocytic or monocytic cell lines tested, vpx mutant virus grew as well as wild-type virus. Only in primary peripheral blood mononuclear cell cultures, severely retarded growth of mutant virus was observed. No replication of vpx-minus virus was detected in primary macrophage cells. A highly sensitive single-round replication assay system was used to determine the defective replication phase in primary mononuclear cells of vpx mutant virus. In all cell lines examined, vpx mutant displayed no abnormality. In contrast, the vpx mutant was demonstrated to be defective at an early stage of the infection cycle in primary cell cultures. No evidence of a replication-defect at a late phase in primary cells of the vpx mutant was obtained by a transfection-coculture method. These results indicate that the virion-associated Vpx protein is essential for early viral replication process in natural target cells such as primary macrophages.
Meiko Kawamura, T Ishizaki, A Ishimoto, T Shioda, T Kitamura and Akio Adachi : Growth ability of human immunodeficiency virus type 1 auxiliary gene mutants in primary blood macrophage cultures, The Journal of General Virology, Vol.75, No.9, 2427-2431, 1994.
(Summary)
A strain of human immunodeficiency virus type 1 that is strictly tropic for primary human blood cell cultures was constructed in vitro. Mutational studies on the vif, vpr, vpu and nef genes of this virus were performed to evaluate their biological functions in natural target cells. For this purpose, replication properties of mutant viruses in peripheral blood mononuclear cells (PBMCs) and macrophages (PBMPs) were determined. Three phenotypes with respect to virus replication were noticed: normal or mildly retarded growth (nef and vpr mutants), impaired growth (vpu mutant), and no growth (vif mutant). These results suggest that the Vif and Vpu proteins are more important than the Nef and Vpr proteins for virus replication in PBMCs and PBMPs.
Tatsuhiko Igarashi, Riri Shibata, Futoshi Hasebe, Yasushi Ami, Katsuaki Shinohara, Toshihiko Komatsu, Christiane Stahl-Henning, Harald Petry, Gerhard Hunsmann, Takeo Kuwata, Minghao Jin, Akio Adachi, Takashi Kurimura, Mineyuki Okada, Tomoyuki Miura and Masanori Hayami : Persistent infection with SIVmac chimeric virus having tat, rev, vpu, env, and nef of HIV-1 in macaque monkeys, AIDS Research and Human Retroviruses, Vol.10, No.8, 1021-1029, 1994.
(Summary)
A chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu, env, and nef genes of human immunodeficiency virus type 1 was generated. The chimeric virus, NM-3n, grew competently in peripheral blood mononuclear cells from cynomolgus monkeys like the parental SIVmac. Two cynomolgus monkeys and one rhesus monkey inoculated with NM-3n raised antibodies to SIVmac Gag and HIV-1 Env. The antibodies raised in the cynomolgus monkeys persisted for at least 1.7 years. The antibodies contained virus neutralizing activity not only to the original chimeric virus but also to the parental HIV-1. Infectious viruses were isolated from one of the cynomolgus monkeys 37 and 63 weeks after inoculation and from the rhesus monkey continuously from 6 weeks after infection onward. The recovered virus maintained its chimeric structure but included several clones with mutations in the env V3 region. When the recovered virus was inoculated to another rhesus monkey, no difference in the frequency of virus recovery was seen from the originally infected monkeys. These carrier monkeys have so far shown no sign of the disease.
Yoshiaki Nakagawa, Kazuhiro Kakimi, Ling Wang, Yoshinao Kubo, Kyoko Higo, Toru Masuda, Kagemasa Kuribayashi, Michihiro Iwashiro, Yosuke Komatz, Toshiyasu Hirama, Akio Adachi and Akinori Ishimoto : Inhibition of murine AIDS(MAIDS) development by the transplantation of bone marrow cells carrying the Fv-4 resistant gene to MAIDS virus-infected mice, Journal of Virology, Vol.68, No.3, 1438-1441, 1994.
187.
Keizo Tomonaga, S Y Shin, M Fukasawa, Takayuki Miyazawa, Akio Adachi and Takeshi Mikami : Feline immunodeficiency virus gene expression: analysis of the RNA splicing pattern and monocistronic rev mRNA, The Journal of General Virology, Vol.74, No.11, 2409-2417, 1993.
(Summary)
The transcription pattern of the feline immunodeficiency virus (FIV) genome in a feline CD4+ cell line was examined. In addition to the genomic RNA (9.2 kb), at least five FIV-specific transcripts [5.2, 4.4 (doublet), 1.7 and 1.4 kb] were detected by using subgenomic restriction enzyme fragments of an FIV molecular clone or FIV-specific oligonucleotides as probes. Among these transcripts, the 9.2, 5.2 and 4.4 (doublet) kb mRNAs were not expressed in the cytoplasm of cells transfected with a rev- mutant. To determine the location of splice junctions in the FIV genome, we used PCR to amplify and clone cDNAs corresponding to the viral mRNAs from infected cells. The region between pol and env was found to contain at least two splice donor and three splice acceptor sites. Two splice acceptor sites were detected in the 3' region of env. By hybridization analysis and sequencing of cDNA clones, it was revealed that the medium sized mRNAs are derived from a single splice event, with different splice acceptor sites, and that the two smaller transcripts are doubly or triply spliced mRNAs. Our results demonstrate a complex pattern of alternative splicing of FIV mRNAs. Furthermore, we identified monocistronic rev mRNA species that employ a unique splice acceptor site.
Keizo Tomonaga, Takayuki Miyazawa, Juni-chi Sakuragi, Takeshi Mori, Akio Adachi and Takeshi Mikami : The feline immunodeficiency virus ORF-A gene facilitates efficient viral replication in established T-cell lines and peripheral blood lymphocytes, Journal of Virology, Vol.67, No.10, 5889-5895, 1993.
(Summary)
Frameshift mutants corresponding to all of the identified open reading frames of feline immunodeficiency virus, including the ORF-A gene, which has an unknown function, were constructed in vitro. Upon transfection into cells, no significant difference between the phenotypes of ORF-A mutant clones and those of wild-type clones was demonstrated. Although only ORF-A mutant virus among all mutant viruses from transfected cells showed infectivity in established T-cell lines, the replication and propagation of the ORF-A mutant virus were efficiently reduced compared with those of the wild-type virus. Moreover, the loss of the function of the ORF-A gene resulted in a severe defect in productive infection in primary peripheral blood lymphocytes both in the amount of reverse transcriptase activity produced and in core protein expression. These findings demonstrate that the ORF-A gene of feline immunodeficiency virus is required for efficient viral replication and suggest that the ORF-A gene is likely to be important for natural infection.
Takayuki Miyazawa, M Kohmoto, Y Kawaguchi, Keizo Tomonaga, T Toyosaki, K Ikuta, Akio Adachi and Takeshi Mikami : The AP-1 binding site in the feline immunodeficiency virus long terminal repeat is not required for virus replication in feline T lymphocytes, The Journal of General Virology, Vol.74, No.8, 1573-1580, 1993.
(Summary)
Sequences of 31 bp containing putative AP-1 and AP-4 binding sequences in the U3 region of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were deleted and the basal promoter activity of the LTR was measured by the chloramphenicol acetyltransferase (CAT) assay. The activity of the FIV LTR was reduced in Felis catus whole foetus 4 (fcwf-4) cells and Crandell feline kidney cells by this deletion. Cotransfection of murine c-Fos or c-Jun expression plasmids with the FIV LTR-CAT reporter plasmid into fcwf-4 cells revealed that FIV LTR could be activated by c-Fos but not c-Jun in the cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the replication rate and the cytopathogenic activity of the mutant were compared with those of the wild-type in two feline CD4-positive T lymphoblastoid cell lines. It was found that the rate and activity of the mutant were almost the same as those of the wild-type. From these data, we conclude that the 31 bp fragment is important for achieving maximal expression of the FIV genome, but not required for the replication of FIV in feline T lymphocytes.
Hiroyuki Sakai, Jun-ichi Sakuragi, Sayuri Sakuragi, Meiko Kawamura and Akio Adachi : Compatibility of tat and rev transactivators in the primate lentiviruses, Archives of Virology, Vol.129, No.1-4, 1-10, 1993.
(Summary)
Primate immunodeficiency viruses carry a unique set of transacting regulator genes, which are essential for viral replication. The exchangeability of these Tat and Rev transactivators derived from viruses of the four major subgroups identified to date was assessed in transient transfection and infection assay systems. The human immunodeficiency virus type 1 (HIV-1), a major causative virus of human AIDS, efficiently activated the other viruses. In contrast, the tat and rev gene products of HIV-2, SIVAGM (virus of the African green monkey), and SIVMND (virus of the mandrill) did not fully transactivate the HIV-1. In particular, the rev of HIV-1 was not substantially replaced by those of the other viruses. The result that HIV-1 is distinct from the other immunodeficiency viruses with respect to the compatibility of two transactivators gives a firm functional basis for the unique phylogenetic position of HIV-1.
(Keyword)
Amino Acid Sequence / Animals / Base Sequence / Gene Products, rev / Gene Products, tat / HIV-1 / Humans / Lentivirus / Molecular Sequence Data / RNA, Viral / rev Gene Products, Human Immunodeficiency Virus / tat Gene Products, Human Immunodeficiency Virus
Hiroyuki Sakai, Riri Shibata, Jun-ichi Sakuragi, Sayuri Sakuragi, Meiko Kawamura and Akio Adachi : Cell-dependent requirement of human immunodeficiency virus type 1 Vif protein for maturation of virus particles, Journal of Virology, Vol.67, No.3, 1663-1666, 1993.
192.
Hiroyuki Sakai, Meiko Kawamura, Jun-ichi Sakuragi, Sayuri Sakuragi, Riri Shibata, Akinori Ishimoto, Nobumi Ono, Sigeharu Ueda and Akio Adachi : Integration is essential for efficient gene expression of human immunodeficiency virus type 1, Journal of Virology, Vol.67, No.3, 1169-1174, 1993.
(Summary)
A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.
H. Sakai, S. Sakuragi, J. Sakuragi, M. Kawamura, R. Shibata and Akio Adachi : Sequences responsible for efficient replication of simian immunodeficiency virus SIVmnd in cells of the monocyte/macrophage lineage, The Journal of General Virology, Vol.73, No.11, 2989-2993, 1992.
(Summary)
We determined the susceptibility of monocytic cell lines to infection with viral strains derived from two infectious clones of simian immunodeficiency virus isolated from a mandrill. One of the strains, which replicates poorly in T cell lines, was found to grow more rapidly than the other in these cells. The viral determinant for this property was genetically mapped within the env gene encoding a surface protein. Six amino acid substitutions identified appeared to be located outside of the domains corresponding to human immunodeficiency virus type 1 env functional domains such as the CD4-binding and V3 loop regions.
(Keyword)
Animals / Base Sequence / Cell Line / Genes, env / Humans / Macrophages / Molecular Sequence Data / Monocytes / Organ Specificity / RNA, Viral / Simian immunodeficiency virus / Virus Replication
S. Sakuragi, R. Shibata, R. Mukai, T. Komatsu, M. Fukasawa, H. Sakai, J. Sakuragi, M. Kawamura, K. Ibuki, M. Hayami and Akio Adachi : Infection of macaque monkeys with a chimeric human and simian immunodeficiency virus, The Journal of General Virology, Vol.73, No.Pt11, 2983-2987, 1992.
(Summary)
Two macaque monkeys were inoculated with a chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu and env genes of human immunodeficiency virus type 1. Infectious virus was recovered from one of the monkeys at 2 and 6 weeks post-infection. The hybrid nature of the isolated viruses was verified by Southern and Western blotting analyses. Both of the monkeys infected with the chimera elicited a humoral antibody response against the virus.
Keizo Tomonaga, Junzo Norimine, Yeon-Sil Shin, Masashi Fukasawa, Takayuki Miyazawa, Akio Adachi, Tomoko Toyosaki, Yasushi Kawaguchi, Chieko Kai and Takeshi Mikami : Identification of a feline immunodeficiency virus gene which is essential for cell-free virus infectivity, Journal of Virology, Vol.66, No.10, 6181-6185, 1992.
(Summary)
Feline immunodeficiency virus (FIV) contains at least three small open reading frames (ORFs) in the genome, in addition to the three structural genes. Two of these ORFs (putative vif and ORF-A) have unknown functions. Northern (RNA) blot analysis of mRNAs from an FIV-infected cell line showed that the putative-vif-specific mRNA was expressed as a 5.2-kb species. To examine the function of the putative vif gene, we constructed mutants carrying a deletion in either the vif-like gene or the rev gene from an infectious molecular clone of FIV. Although the vif mutant produced virion-associated reverse transcriptase at a normal level upon transfection, cell-free virus prepared from the transfected cells could not infect feline CD4+ cells. The infectivity of the vif mutant, however, was demonstrated in a coculture of the transfected cells and feline CD4+ cells. We conclude that FIV contains the vif gene, which is structurally and functionally similar to that of the primate lentiviruses.
(Keyword)
Base Sequence / Blotting, Northern / Cell-Free System / Chromosome Deletion / Genes, Viral / Immunodeficiency Virus, Feline / Molecular Sequence Data / Open Reading Frames / RNA, Viral / RNA-Directed DNA Polymerase / Viral Structural Proteins
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 1382146
Jun-ichi Sakuragi, Hiroyuki Sakai, Sayuri Sakuragi, Riri Shibata, Simon Wain-Hobson, Masanori Hayami and Akio Adachi : Functional classification of simian immunodeficiency virus isolated from a chimpanzee by transactivators, Virology, Vol.189, No.1, 354-358, 1992.
(Summary)
In reporter-based transient expression systems, we characterized simian immunodeficiency virus from a chimpanzee (SIVCPZ), with special reference to the human immunodeficiency virus type 1 (HIV-1). SIVCPZ was not equally activated by tat and rev transactivators derived from representative primate lentiviruses. HIV-1 alone activated SIVCPZ to the full extent in both tat and rev assays. The tat and rev gene products of SIVCPZ, as well as those of HIV-1, efficiently transactivated the other viruses. These results indicate that SIVCPZ is identical to HIV-1 with regard to the compatibility of tat and rev gene activities among four subgroups of primate lentiviruses.
(Keyword)
Amino Acid Sequence / Animals / Base Sequence / Chloramphenicol O-Acetyltransferase / DNA, Recombinant / Gene Expression Regulation, Viral / Gene Products, rev / Gene Products, tat / HIV-1 / Molecular Sequence Data / Nucleic Acid Conformation / Pan troglodytes / RNA, Viral / Sequence Homology, Nucleic Acid / Simian immunodeficiency virus / Trans-Activators / rev Gene Products, Human Immunodeficiency Virus / tat Gene Products, Human Immunodeficiency Virus
Hiroyuki Sakai, Jun-Ichi Sakuragi, Sayuri Sakuragi, Riri Shibata and Akio Adachi : Functional analysis of biologically distinct genetic variants of simian immunodeficiency virus isolated from a mandrill, Virology, Vol.189, No.1, 161-166, 1992.
(Summary)
We examined the biological properties of two infectious clones of a simian immunodeficiency virus, SIVMND, which were designated as pMD121 and pMD122. Upon transfection into CD4-negative cells, pMD122 generated virions much less efficiently than pMD121. Likewise, the growth kinetics in CD4-positive cells of virus derived from pMD122 were remarkably delayed relative to those of virus from pMD121. The cytocidal activity of the MD122 virus was also low. A series of recombinant clones were constructed from pMD121 and pMD122 to determine the sequence responsible for the low virulence of the MD122 virus. The genetic determinant in pMD122 responsible for its properties was mapped to within a region (316 base pairs) encompassing the tat, rev, and env coding sequences. Sequence analysis revealed that the two clones differed by only one nucleotide in this region. A nucleotide substitution G (pMD121) to T (pMD122) altered an arginine codon to a serine codon in the first tat coding exon. Transient transfection experiments showed that the tat activity of pMD122 was about twofold less than that of pMD121. These findings indicate that small differences in tat activity can have a dramatic effect on the biological behavior of SIVMND.
T. Miyazawa, Y. Kawaguchi, M. Kohmoto, J. Sakuragi, Akio Adachi, M. Fukasawa and T. Mikami : Production of feline immunodeficiency virus in feline and non-feline non-lymphoid cell lines by transfection of an infectious molecular clone, The Journal of General Virology, Vol.73, No.Pt 6, 1543-1546, 1992.
(Summary)
An infectious molecular clone of the TM1 strain of feline immunodeficiency virus (FIV) was transfected into each of one feline (CRFK), two simian (COS and Vero) and two human (SW480 and HeLa) non-lymphoid cell lines, and virus production was assayed on feline T lymphoblastoid MYA-1 cells by monitoring reverse transcriptase activity. Infectious virus was produced in CRFK, Vero and HeLa cells, but not in COS and SW480 cells. When the basal promoter activity of the FIV long terminal repeat (LTR) was examined in these cell lines by using a chloramphenicol acetyltransferase assay, the activity correlated with the virus production in each cell line. Furthermore, when the activity of the FIV LTR was compared with those of three primate lentivirus LTRs, the highest activity in all the cell lines examined was produced by the LTR of simian immunodeficiency virus from African green monkey (SIVAGM), suggesting that it has a wide expression range. In COS and SW480 cells, the activity of the FIV LTR was much lower than that of the SIVAGM LTR. These results indicate that, whereas the primary block to FIV infection of certain cells may occur at the cell surface, the FIV LTR may also participate in controlling virus replication, as an intracellular mechanism.
(Keyword)
Animals / Cats / Cell Line / Cloning, Molecular / HeLa Cells / Humans / Immunodeficiency Virus, Feline / Kinetics / Transfection / Vero Cells / Virus Replication
H. Sakai, J. Sakuragi, S. Sakuragi, R. Shibata, M. Hayami, A. Ishimoto and Akio Adachi : Genetic characterization of simian immunodeficiency virus isolated from an African mandrill, Archives of Virology, Vol.125, No.1-4, 1-14, 1992.
(Summary)
We constructed an infectious molecular clone of simian immunodeficiency virus from an African mandrill (SIVMND). Upon transfection, this clone directed the production of progeny virus particles infectious to and cytopathic for CD4+ human leukemia cells. Thirteen frameshift proviral mutants with an alteration in the eight open reading frames of SIVMND were generated by recombinant DNA techniques, and were analyzed biologically and biochemically. While mutations in the structural genes gag, pol, and env abolished viral growth and induction of cytopathology, mutants of the vif, vpr, and nef genes were fully biologically active. Of the tat and rev mutants, only one rev mutant grew in CD4+ cells with delayed kinetics. In reporter-based transient expression systems, transactivation potentials of the tat and rev mutants were evaluated. A mutant lacking 2nd coding exon of tat gene exhibited tat activity similar to that of the wild type clone. The infectious rev mutant was partially defective for rev gene activity.
Jun-Ichi Sakuragi, Masashi Fukasawa, Riri Shibata, Hiroyuki Sakai, Meiko Kawamura, Hirofumi Akari, Takahiro Kiyomasu, Akinori Ishimoto, Masanori Hayami and Akio Adachi : Functional analysis of long terminal repeats derived from four strains of simian immunodeficiency virus SIVagm in relation to other primate lentiviruses, Virology, Vol.185, No.1, 455-459, 1991.
(Summary)
The promoter activity of long terminal repeats (LTRs) of four strains of the simian immunodeficiency virus isolated from African green monkeys (SIVAGM) was compared with those of various LTRs derived from the other representative primate lentiviruses: human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2), SIV from a rhesus monkey (SIVMAC), and SIV from a mandrill (SIVMND). The expression of the LTRs was evaluated by monitoring chloramphenicol acetyltransferase production after transfection of reporter plasmid clones. In the absence of viral tat, all SIVAGM LTRs acted as much more efficient promoters than any of the other LTRs. When tat gene products were supplied in trans, LTRs of SIVAGM and SIVMND were activated inefficiently relative to high responder LTRs of HIV-2 and SIVMAC. The LTR of HIV-1 was highly activated by HIV-1 tat, but not so much by HIV-2, SIVAGM, and SIVMND tat.
Hiroyuki Sakai, Riri Shibata, Jun-Ichi Sakuragi, Takahiro Kiyomasu, Meiko Kawamura, Masanori Hayami, Akinori Ishimoto and Akio Adachi : Compatibility of rev gene activity in the four groups of primate lentiviruses, Virology, Vol.184, No.2, 513-520, 1991.
(Summary)
The compatibility of rev genes derived from various primate immunodeficiency viruses of all distinct subgroups identified was assessed in three experimental systems: complementation experiments between proviral rev and gag mutants, evaluation of the ability of the rev gene products to activate proviral reporters, and examination of the capacity of various viruses to augment marker gene expression in the infected reporter cell lines. In all systems, human immunodeficiency virus type 1 (HIV-1) rev was not substantially substituted or was extremely poorly substituted by the rev of the other viruses. The rev of simian immunodeficiency virus (SIV) from a mandrill could be exchanged by HIV-1 rev. In contrast, the rev gene products of all viruses efficiently activate HIV-2 and SIV from an African green monkey.
(Keyword)
Base Sequence / Cell Line / Cloning, Molecular / Gene Expression Regulation, Viral / Genes, gag / Genes, rev / Genetic Complementation Test / HIV-1 / HIV-2 / Lentivirus / Molecular Sequence Data / Mutation / RNA, Viral / RNA-Directed DNA Polymerase / Simian immunodeficiency virus
Takahiro Kiyomasu, Takayuki Miyazawa, Tetsuya Furuya, Riri Shibata, Hiroyuki Sakai, Jun-ichi Sakuragi, Masashi Fukasawa, Noboru Maki, Akira Hasegawa, Takeshi Mikami and Akio Adachi : Identification of the feline immunodeficiency virus rev gene activity, Journal of Virology, Vol.65, No.8, 4539-4542, 1991.
(Summary)
We constructed 16 deletion mutants from an infectious molecular clone of feline immunodeficiency virus (FIV) and a reporter plasmid carrying the bacterial chloramphenicol acetyltransferase (CAT) gene to identify the rev transactivator activity of the virus. Cotransfections of various mutants and the rev reporter clone bearing a portion of FIV env in addition to the CAT gene revealed that the sequence important for the augmentation of CAT production was located in three separate parts of the virus genome. This enhancement was FIV specific in that the human retrovirus rev and rex gene products did not activate the reporter. The phenotypic properties of an FIV proviral mutant containing a small deletion in the genome were similar to those of rev mutants derived from primate immunodeficiency viruses. These results indicate that FIV, like the other lentiviruses, contains the rev gene in its genome.
Riri Shibata, Meiko Kawamura, Hiroyuki Sakai, Masanori Hayami, Akinori Ishimoto and Akio Adachi : Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells, Journal of Virology, Vol.65, No.7, 3514-3520, 1991.
(Summary)
We constructed five chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVMAC) and four SIVMAC mutants by recombinant DNA techniques. Three chimeric clones and all mutants with an alteration in either the vif, vpx, vpr, or nef gene were infectious to human CD4-positive cell lines. The susceptibility of macaque monkey peripheral blood mononuclear cells (PBMC) to infection by these mutants and chimeras was examined in vitro. Macaque PBMC supported the replication of wild-type and vpx, vpr, and nef mutant SIVMAC strains. A chimera carrying the long terminal repeats (LTRs), gag, pol, vif, and vpx of SIVMAC and tat, rev, vpu, and env of HIV-1 was also replication competent in PBMC. In contrast, HIV-1, the vif mutant of SIVMAC, a chimera containing rev and env of SIVMAC, and a chimera containing vpx, vpr, tat, rev, and env of SIVMAC did not grow in PBMC. Western immunoblotting analysis of the replicating chimera in PBMC confirmed the hybrid nature of the virus. These data strongly suggested that the sequence important for macaque cell tropism lies within the LTR, gag, pol, and/or vif sequences of the SIVMAC genome.
Akio Adachi, N. Ono, H. Sakai, R. Shibata, K. Ogawa, T. Kiyomasu, H. Masuike and S. Ueda : Generation and characterization of the human immunodeficiency virus type 1 mutants, Archives of Virology, Vol.117, No.1-2, 45-58, 1991.
(Summary)
Mutations were introduced by recombinant DNA techniques into 9 genes of an infectious molecular clone of human immunodeficiency virus type 1. The 24 mutants generated were characterized biochemically and biologically by transfection and infection experiments. None of the mutants which have mutations in gag (p17, p24, and p15 regions), pol (protease, reverse transcriptase, and endonuclease domains), env (gp120 region), tat, or rev were infectious, whereas vif, vpr, vpu, some of env (gp41) and nef mutants could grow in human CD4+ cells to various degrees. Of the non-infectious mutants, only endonuclease (pol) and gp41 mutants exhibited normal phenotypes with respect to the production of functional reverse transcriptase, the expression of gag, pol, and env proteins, and the generation of progeny virions, when examined in transient assays. All infectious mutants killed the CD4+ cells with the exception of a mutant carrying a defect in the vif gene.
Y. Nishino, M. Kishi, M. Sumiya, K. Ogawa, Akio Adachi, K. Maotani-Imai, S. Kato, K. Hirai and K. Ikuta : Human immunodeficiency virus type 1 vif, vpr and vpu mutants can produce persistently infected cells, Archives of Virology, Vol.120, No.3-4, 181-192, 1991.
(Summary)
A series of human immunodeficiency virus type 1 (HIV-1) mutants in vif, vpr, vpu, and nef were constructed from an infectious plasmid (pNL 432) containing the full-length HIV-1 DNA by frameshift mutations. The capacities for replication and cell killing of these mutant viruses were examined in a clonal cell line (M 10) isolated from HTLV-I-transformed MT-4 cells. In all cases, the mutant viruses replicated, expressed HIV-1 antigens, and induced drastic cytopathic effects. However, some M 10 cells survived infection with vif, vpr, and vpu mutant viruses and became persistently HIV-1-infected, whereas no cells survived infection with the nef mutant as well as the wild-type virus. The HIV-1 particles produced from the surviving cells after infection with the vif, vpr, or vpu mutant viruses were fully replicative in M 10 cells without apparent cytopathic effects.
Riri Shibata, Hiroyuki Sakai, Takahiro Kiyomasu, Akinori Ishimoto, Masanori Hayami and Akio Adachi : Generation and characterization of infectious chimeric clones between human immunodeficiency virus type 1 and simian immunodeficiency virus from an African green monkey, Journal of Virology, Vol.64, No.12, 5861-5868, 1990.
(Summary)
A series of chimeric clones of human immunodeficiency virus type 1 and simian immunodeficiency virus isolated from an African green monkey was constructed in vitro. In transient transfection experiments, all clones produced virion-associated reverse transcriptase, gag proteins, and env proteins. Eight out of 10 chimeric viruses clearly grew in the human CD4+ cell line C8166. Susceptibility of other CD4+ cell lines, MT-4, A3.01, and Molt4 clone 8, to infection with these viruses was also demonstrated.
Hiriyuki Sakai, Haruhiko Siomi, Hisatoshi Shida, Riri Shibata, Takahiro Kiyomasu and Akio Adachi : Functional comparison of transactivation by human retrovirus rev and rex genes, Journal of Virology, Vol.64, No.12, 5833-5839, 1990.
(Summary)
The effect of rev-responsive element deletion on human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) gene expression was examined. The phenotypes of HIV-1 and HIV-2 provirus DNAs lacking the rev-responsive element, as determined by transfection experiments, were indistinguishable from those of virus DNAs carrying rev gene mutations. By using rev-response elements derived from these two viruses, we developed two monitoring systems to evaluate the functionality of HIV-1 rev, HIV-2 rev, and human T-lymphotropic virus type I rex. In both systems, HIV-1 rev and human T-lymphotropic virus type I rex transactivated HIV-2 very efficiently. On the contrary, HIV-2 rev and human T-lymphotropic virus type I rex were poor activators of HIV-1. No functional replacement of rex by HIV-2 rev was observed.
Akihiko Sato, Hisanaga Igarashi, Akio Adachi and Masanori Hayami : Identification and localization of the vpr gene product of human immunodeficiency virus type 1, Virus Genes, Vol.4, No.4, 303-312, 1990.
(Summary)
The entire vpr gene of human immunodeficiency virus type 1 (HIV-1) was cloned into procaryotic and eucaryotic expression vectors. Production of authentic protein encoded by the gene in bacterial and mammalian cells was monitored by Western blotting using guinea pig antisera raised against an N-terminal 14-oligopeptide of the predicted vpr protein. A specific 12-kD protein was clearly detected with these antisera, but not with preimmune sera, in both cell systems, and this binding was blocked by the oligopeptide. These antisera also recognized a protein of the same size in several human T-cell lines infected with HIV-1. Western blotting analysis of subcellular fractions prepared from the cells producing wildtype vpr protein strongly suggested that the protein was membrane associated. A region within the vpr required for the stable expression of vpr product was also suggested by mutational analyses.
(Keyword)
Amino Acid Sequence / Animals / Blotting, Western / Cell Line / Cloning, Molecular / Cricetinae / DNA Mutational Analysis / DNA, Viral / gene expression / Gene Products, vpr / HIV-1 / Molecular Sequence Data / T lymphocytes / Transformation, Genetic / Tumor Cells, Cultured / vpr Gene Products, Human Immunodeficiency Virus
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 2149621
Kazuhiro Kakimi, Yoshiko Kishida, Ikuko Higuchi, Takahiro Kiyomasu, Hiroyuki Sakai, Riri Shibata, Shin-ichiro Yanagawa, Akio Adachi and Akinori Ishimoto : Fv-1 restriction of endogenous feline C-type RD114 virus genome phenotypically mixed with ecotropic murine leukemia viruses, Japanese Journal of Cancer Research, Vol.81, No.8, 768-772, 1990.
(Summary)
Endogenous feline leukemia RD114 virus genome rendered capable of infecting mouse cells by phenotypic mixing with an ecotropic murine leukemia virus (MuLV) exhibited the Fv-1 restriction pattern of the ecotropic murine virus. However, RD114 genomes phenotypically mixed with ecotropic MuLV showed one-hit dose-response kinetics, even when titrated with murine cells with the restricted Fv-1 phenotype.
Atsushi Saitoh, Hiroshi Iwasaki, Atsuo Nakata, Akio Adachi and Hideo Shinagawa : Overproduction of human immunodeficiency virus type I reverse transcriptase in Escherichia coli and purification of the enzyme, Microbiology and Immunology, Vol.34, No.6, 509-521, 1990.
(Summary)
Overexpression of the reverse transcriptase was designed in E. coli. For a high level of expression, HIV protein was expressed as a protein fusion with beta-galactosidase. When the proviral DNA fragment covering the 3' half of the gag gene and the entire pol gene was ligated to the 3' end of the lacZ gene to fuse the truncated gag to lacZ in frame, a small quantity of reverse transcriptase was produced, indicating that frameshifting and post-translational processing have occurred. Much more reverse transcriptase was produced when the entire pol region was directly fused to the lacZ gene. From a one liter culture of bacteria, 1 mg of highly purified reverse transcriptase consisting of approximately equimolar amounts of two species (p64 and p51) was obtained. These proteins had identical N-termini consistent with the deduced amino acid sequence and therefore, might be correctly processed from the fusion protein in E. coli by the protease encoded by the pol region. The purified reverse transcriptase was enzymatically as active as the enzyme purified from the virus particles, and immunoreactive to the sera of HIV carriers with high sensitivity and specificity.
(Keyword)
Cloning, Molecular / Electrophoresis, Polyacrylamide Gel / Endopeptidases / Escherichia coli / HIV Antibodies / HIV Infections / HIV-1 / Humans / Immunoblotting / Plasmids / Protein Biosynthesis / Protein Processing, Post-Translational / RNA-Directed DNA Polymerase / Recombinant Fusion Proteins / beta-Galactosidase
Hiroyuki Sakai, Riri Shibata, Tomoyuki Miura, Masanori Hayami, Koji Ogawa, Takahiro Kiyomasu, Akinori Ishimoto and Akio Adachi : Complementation of the rev gene mutation among human and simian lentivuruses, Journal of Virology, Vol.64, No.5, 2202-2207, 1990.
(Summary)
The functional exchangeability of the rev gene was assessed in transient transfection experiments by using in vitro-constructed rev and gag mutants of the following three primate lentiviruses: human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus from the African green monkey (SIV AGM). Cotransfection into SW480 cells of the rev and gag mutants derived from the DNA of each infectious virus resulted in the generation of progeny particles as determined by reverse transcriptase assay. rev gene mutants of HIV-2 and SIV AGM were also complemented by all gag mutants derived from the three viruses. In contrast, no evidence of complementation was obtained following cotransfection of the HIV-1 rev mutant and the gag mutant of HIV-2 or SIV AGM.
M. Nishimura, Akio Adachi, M. Maeda, A. Ishimoto and J. Kimura : Human T lymphotropic virus type I may not be associated with multiple sclerosis in Japan, The Journal of Immunology, Vol.144, No.5, 1684-1688, 1990.
213.
M. Nishimura, Akio Adachi, I. Akiguchi, N. Shirahata, M. Maeda, A. Ishimoto, T. Mezaki and J. Kimura : High ratio of HTLV-1-infected cells in patients with HTLV-1 associated myelopathy(HAM), Acta Neurologica Scandinavica, Vol.81, No.3, 209-214, 1990.
(Summary)
Sixteen patients with HTLV-1 associated myelopathy (HAM) were examined for the presence of HTLV-1 provirus genome by Southern blot analysis of genomic DNA from peripheral blood mononuclear (PBM) cells. Random integration of the provirus was detected in 14 of 16 HAM patients. By contrast, the provirus genome could not be detected in 6 non-HAM HTLV-1 carriers, HAM patients were found to have significantly higher antibody titer to HTLV-1 in the sera compared with carriers. These features of HAM patients, i.e., detectable levels of provirus integration in PBM cells and high antibody titer to HTLV-1 in the sera, were noted in 2 wives of HAM patients with neurological signs and abnormalities. High anti-HTLV-1 antibody titer and detection of the provirus genome by Southern hybridizations may be useful for screening subclinical HAM cases and elucidating pathogenesis.
Riri Shibata, Tomoyuki Miura, Masanori Hayami, Koji Ogawa, Hiroyuki Sakai, Takahiro Kiyomasu, Ashimoto Ishimoto and Akio Adachi : Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIVagm, Journal of Virology, Vol.64, No.2, 742-747, 1990.
(Summary)
We constructed an infectious molecular clone of the human immunodeficiency virus type 2 (HIV-2) and generated nine frameshift mutants corresponding to nine open reading frames identified so far. Three structural (gag, pol, env) and two regulative (tat, rev) gene mutants were not infectious, whereas vif, vpx, vpr, and nef genes were dispensable for infectivity. All of the mutants except env and rev were cytopathic in CD4+ human leukemia cells. In transfection assays, the expression of HIV-2 long terminal repeat was activated by infectious clones of HIV-1, HIV-2, and simian immunodeficiency virus from African green monkey but not by the tat mutants. However, an HIV-2 tat mutant could produce small amounts of virus proteins and particles in contrast to a rev mutant, which directed no detectable synthesis of virus proteins and virions.
Riri Shibata, Tomoyuki Miura, Masanori Hayami, Hiroyuki Sakai, Koji Ogawa, Takahiro Kiyomasu, Akinori Ishimoto and Akio Adachi : Construction and characterization of an infectious DNA clone and of mutants of simian immunodeficiency virus isolated from the African green monkey, Journal of Virology, Vol.64, No.1, 307-312, 1990.
(Summary)
We constructed a full-length molecular clone of simian immunodeficiency virus from an African green monkey. Upon transfection, this clone directed the production of virus particles cytopathic and infectious to human CD4+ leukemia cell lines. Mutations were introduced by recombinant DNA techniques into eight open reading frames of simian immunodeficiency virus from the African green monkey thus far identified. The phenotypes of mutant viruses, i.e., infectivity, cytopathogenicity, transactivation of gene expression controlled by a long terminal repeat, and viral RNA and protein syntheses, were examined by transfection and infection experiments. Three structural (gag, pol, and env) and two regulatory (tat and rev) gene mutants were not infectious, whereas vif, vpx, and nef were dispensable for infectivity and mutant viruses were highly cytopathic. In transient transfection assays, a rev mutant produced mainly small mRNA species and no detectable virus protein and particles. The transactivation potential of a tat mutant was about 10-fold less than that of wild-type DNA, generating small amounts of virus.
R. Shibata, H. Sakai, K. Ogawa, A. Ishimoto and Akio Adachi : Comparative studies on tat mutants of three primate lentiviruses, Archives of Virology, Vol.114, No.3-4, 243-250, 1990.
(Summary)
A frame-shift tat gene mutant of human immunodeficiency virus type 1 (HIV-1), which showed no detectable trans-activation potential, was constructed in vitro. Upon transfection, this clone directed the synthesis of virus mRNAs, gag proteins and virion-associated reverse transcriptase (RT) at a low level as was observed with the tat mutants of HIV-2 and simian immunodeficiency virus isolated from African green monkey (SIVAGM). Using these mutant viruses, trans-activation efficiency of viral gene expression by tat was compared among HIV-1, HIV-2, and SIVAGM. SIVAGM seemed to be less dependent on tat for RT production than HIV-1 and HIV-2.
Sei-Ichi Endo, Satoshi Kubota, Haruhiko Siomi, Akio Adachi, Stephen Oroszlan, Masatoshi Maki and Masakazu Hatanaka : A region of basic-amino-acid-cluster in HIV-1 tat protein is essential for trans-acting activity and nucleolar localization, Virus Genes, Vol.3, No.2, 99-110, 1989.
(Summary)
The trans-acting factor of human immunodeficiency virus (HIV), Tat, has a basic amino-acid cluster that is highly conserved among different HIV isolates. We have examined the effects of mutations in the basic region of Tat on its trans-acting activity and cellular localization. Introduction of a stop codon immediately preceding the basic region abolished the activity, while the truncated mutant with the basic region retained some activity. The basic region of Tat was replaceable with that of Rev (another trans-acting factor of HIV) but not with that of adenovirus Ela nor cellular enzyme. The result of immunofluorescence analysis revealed a correlation between the nuclear, especially nucleolar, accumulation and the activities of mutant Tat proteins.
(Keyword)
Amino Acid Sequence / Chromosome Mapping / Cluster Analysis / Fluorescent Antibody Technique / Gene Products, tat / HIV-1 / Molecular Sequence Data / Mutation / Nucleolus Organizer Region / Trans-Activators / Transcriptional Activation / tat Gene Products, Human Immunodeficiency Virus
Koji Ogawa, Riri Shibata, Takahiro Kiyomasu, Ikuko Higuchi, Yoshihiro Kishida, Akinori Ishimoto and Akio Adachi : Mutational analysis of the human immunodeficiency virus vpr open reading framev, Journal of Virology, Vol.63, No.9, 4110-4114, 1989.
(Summary)
Mutations were introduced by recombinant DNA techniques into the vpr open reading frame of an infectious molecular clone of human immunodeficiency virus type 1. The effect of these changes on the replicative and cytopathologic properties of the virus recovered from transfected cells was studied in several human CD4+ lymphocyte cell lines. In all cases, mutant viruses were infectious and cytopathic. However, when a low-input dose was used, mutants grew significantly more slowly than the wild-type virus. The growth kinetics of vpr mutants were distinct from those of vif and vpu mutants.
(Keyword)
Cytopathogenic Effect, Viral / Genes / Genes, Viral / HIV / Mutation / Phenotype / RNA-Directed DNA Polymerase
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 2474678
John Leonard, Jaspal S. Khillan, Howard E. Gendelman, Akio Adachi, Shaun Lorenzo, Heiner Westphal, Malcom A. Martin and Monte S. Meltzer : The human immunodeficiency virus long terminal repeat is preferentially expressed in Langerhans cells in transgenic mice, AIDS Research and Human Retroviruses, Vol.5, No.4, 421-430, 1989.
(Summary)
Four lines of transgenic mice containing the HIV LTR linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT) were constructed. In each line, a characteristic tissue pattern of CAT expression was observed with detectable levels present in the eye, heart, spleen, thymus, and tail. Low levels of CAT were present in circulating lymphocytes, but CAT activity in these cells could be augmented following treatment with the mitogen phytohemagglutinin (PHA). Likewise, CAT expression was present at only low levels in circulating monocytes, but higher levels of CAT were observed in macrophages grown in the presence of various cytokines (CSF-1, GM-CSF, IL-1 alpha, IL-4, and IL-2). Furthermore, Langerhans cells recovered from skin showed higher levels of CAT activity than those observed in other cells of monocyte-macrophage lineage. These results indicate that LTR-CAT expression in cells of monocyte-macrophage lineage may increase in proportion to the degree of differentiation of these cells. These animals may be useful in the study of cell-specific determinants of LTR-directed gene activity and may serve to identify exogenous cofactors that promote the progression of HIV-related disease in vivo.
(Keyword)
Animals / Chloramphenicol O-Acetyltransferase / Gene Products, tat / HIV / Langerhans Cells / Macrophages / Mice / Mice, Transgenic / Repetitive Sequences, Nucleic Acid / T lymphocytes / Transcription Factors / tat Gene Products, Human Immunodeficiency Virus
Masataka Nishimura, Akio Adachi, Michiyuki Maeda, Ichiro Akiguchi, Masashi Fujita, Masakuni Kameyama and Akinori Ishimoto : Analysis of the provirus genome integrated in T cell lines establishied from the cerebrospinal fluid of patients with human T lymphotropic virus type 1-associated myelopathy, Journal of Neuroimmunology, Vol.20, No.1, 33-37, 1988.
(Summary)
T cell lines were established from the cerebrospinal fluid (CSF) lymphocytes of three patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM). To elucidate the possible changes of the provirus nucleotide sequences integrated in HAM-derived T cell line cells, DNA from these cells was digested with PstI, which cleaved the provirus genome of HTLV-I at several sites, and three common bands were detected by Southern blot analysis in all cases. These bands were identical in size to those detected in T cell lines established from peripheral blood lymphocytes of adult T cell leukemia (ATL) patients. These results were confirmed with restriction enzymes HindIII and BamHI. These findings suggest that the provirus genome detected in T cell lines derived from CSF of HAM patients is identical to HTLV-I.
Gendelman E. Howard, Theodore S. Theodore, Willey Ronald, McCoy John, Akio Adachi, Mervis J. Robert, Venkatesan Sundrarajan and Martin A. Malcolm : Molecular characterization of a polymerase mutant human immunodeficiency virus, Virology, Vol.160, No.2, 323-329, 1987.
(Summary)
A cell line (8E5) containing a single defective copy of human immunodeficiency virus proviral DNA and producing noninfectious viral particles lacking reverse transcriptase (RT) and endonuclease proteins has recently been described (Folks, et al., (1986b) J. Exp. Med. 164, 280-290). In this report, the mutation in a full-length molecular clone of the provirus (p8E5) was mapped to a 1931-bp region of the pol gene encoding RT. The nucleotide sequence of this segment revealed a 1-base deletion 301 codons from the common amino terminus of the 64- and 51-kDa RTs. Expression of the p8E5 RT segment in Escherichia coli generated an enzymatically inactive and truncated 33-kDa protein.
(Keyword)
Amino Acid Sequence / Base Sequence / Cell Line / Cloning, Molecular / Defective Viruses / Escherichia coli / Genes / Genes, Viral / HIV / Molecular Sequence Data / Mutation / RNA-Directed DNA Polymerase / Retroviridae Proteins
Akinori Ishimoto, Masato Takimoto, Akio Adachi, Masahiro Kakuyama, Shigehisa Kato, Kazuhiro Kakimi, Katsuko Fukuoka, Toshiaki Ogiu and Mutsushi Matsuyama : Sequences responsible for erythroid and lymphoid leukemia in the long terminal repeats of Friend-MCF and Moloney murine leukemia viruses, Journal of Virology, Vol.61, No.6, 1861-1866, 1987.
(Summary)
Despite the high degree of homology (91%) between the nucleotide sequences of the Friend-mink cell focus-forming (MCF) and the Moloney murine leukemia virus (MuLV) genomic long terminal repeats (LTRs), the pathogenicities determined by the LTR sequences of the two viruses are quite different. Friend-MCF MuLV is an erythroid leukemia virus, and Moloney MuLV is a lymphoid leukemia virus. To map the LTR sequences responsible for the different disease specificities, we constructed nine viruses with LTRs recombinant between the Friend-MCF and Moloney MuLVs. Analysis of the leukemia induced with the recombinant viruses showed that a 195-base-pair nucleotide sequence, including a 75-base-pair nucleotide Moloney enhancer, is responsible for the tissue-specific leukemogenicity of Moloney MuLV. However, not only the enhancer but also its downstream sequences appear to be necessary. The Moloney virus enhancer and its downstream sequence exerted a dominant effect over that of the Friend-MCF virus, but the enhancer sequence alone did not. The results that three of the nine recombinant viruses induced both erythroid and lymphoid leukemias supported the hypothesis that multiple viral genetic determinants control both the ability to cause leukemia and the type of leukemia induced.
Akio Adachi, Scott Koenig, Howard E. Gendelman, Daryl Daugherty, Sebastiano Gattoni-Celli, Anthony S. Fauci and Malcom A. Martin : Productive, persistent infection of human colorectal cell lines with human immunodeficiency virus, Journal of Virology, Vol.61, No.1, 209-213, 1987.
(Summary)
Thirteen adherent human non-lymphocyte cell lines were tested for their susceptibility to infection by human immunodeficiency virus. Productive infection could be demonstrated in three of five colorectal carcinoma cell lines examined; the other eight human non-lymphocyte cell lines were uninfectible. A susceptible colon carcinoma cell line (HT29), as well as normal colonic mucosa, was shown to contain a 3.0-kilobase species of poly(A)+ CD4 RNA, whereas uninfectible colon carcinoma and rhabdomyosarcoma cell lines synthesized no detectable T4 RNA. A persistently infected colon carcinoma cell line was established that continued to produce progeny human immunodeficiency virus for more than 10 weeks postinfection.
Howard E. Gendelman, Willam Phelps, Lionel Feigenbaum, Jeffrey M. Ostrove, Akio Adachi, Peter M. Howley, George Khoury, Harold S. Ginsberg and Malcom A. Martin : Trans-activation of the human immunodeficiency virus long terminal repeat sequence by DNA viruses, Proceedings of the National Academy of Sciences of the United States of America, Vol.83, No.24, 9759-9763, 1986.
(Summary)
To investigate whether DNA viruses can augment gene expression of the human immunodeficiency virus (HIV), cotransfection experiments were carried out in which a recombinant plasmid containing the HIV long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene was transfected into cultured cells along with plasmids containing DNA from various distinct classes of DNA viruses. Molecular clones containing JC virus, BK virus, lymphotropic papovavirus, bovine papilloma virus, type 1 herpes simplex virus (HSV-1), and varicella-zoster virus sequences increased CAT expression directed by the HIV LTR. Trans-activation of the HIV LTR varied in different cell lines, but in each case the HIV tat gene product elicited the greatest stimulation. Primer-extension assays specific for HIV LTR mRNA revealed increased levels of steady-state RNA following transfection with HIV tat as well as with several of the DNA viruses. Virus-specific RNA expression paralleled the stimulation of CAT activity. More-than-additive effects were observed at both the RNA and protein levels when tat plus type 1 herpes simplex virus DNAs or tat plus JC virus DNAs were transfected into cells with the HIV LTR-CAT plasmid. These data suggest that coinfection of cells by HIV and some DNA viruses can stimulate the expression of HIV.
(Keyword)
Antigens, Viral, Tumor / Cell Line / DNA Viruses / Gene Expression Regulation / HIV / Humans / Promoter Regions, Genetic / RNA, Viral / RNA-Directed DNA Polymerase / Repetitive Sequences, Nucleic Acid / Species Specificity / Transcription, Genetic
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 2432602
Akio Adachi, Howard E. Gendelman, Scott Koenig, Thomas Folks, Ronald Willey, Arnold Rabson and Malcolm A. Martin : Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone, Journal of Virology, Vol.59, No.2, 284-291, 1986.
(Summary)
We constructed an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified.
Akinori Ishimoto, Akio Adachi, Koji Sakai and Mutsushi Matsuyama : Long terminal repeat of Friend-MCF virus contains the sequence responsible for erythroid leukemia., Virology, Vol.141, No.1, 30-42, 1985.
(Summary)
Friend-MCF virus induces erythroid leukemia when injected into newborn NFS mice whereas Moloney virus induces T-cell lymphoma. To identify the portion of Friend-MCF virus responsible for erythroid leukemia induction four in vitro recombinant viruses were constructed in which env regions or U3 regions of LTR were reciprocally exchanged between Friend-MCF and Moloney viruses. A FrMCF-Mol (LTR) virus whose genome was derived primarily from Friend-MCF virus together with 621 nucleotides of Moloney virus at its 3' end including the U3 region of LTR was a thymic lymphoma-inducing virus. A Mol-FrMCF (LTR) virus with the genome derived primarily from Moloney virus but 596 nucleotides of Friend-MCF virus information at the same region as FrMCF-Mol (LTR) was an erythroid leukemia-inducing virus. A Mol-FrMCF (env) virus whose genome was derived primarily from Moloney virus but which had 2.3 kbp of Friend MCF at the 3' end of the pol gene including most of the env gene with all of gp70 and the N terminal of p15E was a lymphoid leukemia-inducing mink cell focus-inducing virus. FrMCF-Mol (env) virus whose genome was derived primarily from Friend-MCF virus but had 2.7 kbp of Moloney virus at the same region as Mol-FrMCF (env) virus was an erythroid leukemia-inducing ecotropic virus. The Mol-FrMCF (LTR) and Mol-FrMCF (env) viruses induced mixed leukemia of erythroid and lymphoid cells in some mice.
Akio Adachi, Koji Sakai, Naomi Kitamura, Shigetada Nakanishi, Ohtsura Niwa, Mutsushi Matsuyama and Akinori Ishimoto : Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA., Journal of Virology, Vol.50, No.3, 813-821, 1984.
(Summary)
The highly oncogenic erythroleukemia-inducing Friend mink cell focus-inducing (MCF) virus was molecularly cloned in phage lambda gtWES.lambda B, and the DNA sequences of the env gene and the long terminal repeat were determined. The nucleotide sequences of Friend MCF virus and Friend spleen focus-forming virus were quite homologous, supporting the hypothesis that Friend spleen focus-forming virus might be generated via Friend MCF virus from an ecotropic Friend virus mainly by some deletions. Despite their different pathogenicity, the nucleotide sequences of the env gene of Friend MCF virus and Moloney MCF virus were quite homologous, suggesting that the putative parent sequence for the generation of both MCF viruses and the recombinational mechanism for their generation might be the same. We compare the amino acid sequences in lymphoid leukemia-inducing ecotropic Moloney virus and Moloney MCF virus, and erythroblastic leukemia-inducing ecotropic Friend virus, Friend-MCF virus, and Friend spleen focus-forming virus. The Friend MCF virus long terminal repeat was found to be 550 base pairs long. This contained two copies of the 39-base-pair tandem repeat, whereas the spleen focus-forming virus genome contained a single copy of the same sequence.
Akio Adachi, Koji Sakai and Akinori Ishimoto : Isolation and characterization of mink cell focus-inducing murine leukemia viruses with xenotropic host range from mouse strain SL., Journal of Virology, Vol.45, No.1, 447-451, 1983.
229.
Koji Sakai, Hiroshi Narita, Akio Adachi, Satoru Tsuruta, TToru Yorifuji and Akinori Ishimoto : Fv-1 determinants in xenotropic murine leukemia viruses studied with biological assay systems: isolation of xenotropic virus with N-tropic Fv-1 activity in the cryptic form., Journal of Virology, Vol.42, No.1, 331-336, 1982.
(Summary)
By a biological assay system using phenotypically mixed ecotropic and xenotropic murine leukemia viruses, we investigated whether in the virions of a xenotropic virus there is N- or B-tropic Fv-1 determinant in active form. The existence of N-tropic Fv-1 determinant was demonstrated in SL-XT-1 xenotropic virus isolated from the spleen of a 3-month-old SL mouse, and the N-tropic Fv-1 tropism was confirmed by analysis of the phenotypically mixed viruses harvested from clonal SC-1 cells doubly infected with the SL-XT-1 and B-tropic ecotropic viruses. However, neither N- nor B-tropic Fv-1 determinant was demonstrated in any xenotropic viruses isolated from embryo cells of BALB/c, NZB, or DBA/2 mice, or Cas E #1-IU, and xenotropic-like virus isolated from a wild mouse.
Hiroshi Shibuta, Akio Adachi, Tadahito Kanda and Minoru Matumoto : Experimental parainfluenzavirus infection in mice: fatal illness with atrophy of thymus and spleen in mice caused by a variant of parainfluenza 3 virus, Infection and Immunity, Vol.35, No.2, 437-441, 1982.
Virus clones lacking detectable neuraminidase activity (SC-YN and M-YN) as well as those possessing it (LT-910N and LT-YN) were isolated from bovine strains of parainfluenza 3 virus. LT-910N and LT-YN viruses produced large turbid plaques in MDBK cells, and SC-YN virus produced small clear plaques. Incorporation of a bacterial neuraminidase in agar overlay medium made SC-YN virus form large turbid plaques, whereas it made M-YN virus form large clear plaques. However, M-YN virus formed only pinhole plaques or no plaques in the absence of neuraminidase. The exogenous neuraminidase had little effect on the plaque formation of LT-910N and LT-YN viruses. M-YN virus induced extensive syncytial formation, and SC-YN virus produced less extensive syncytial formation. The exogenous neuraminidase enhanced replication of SC-YN and M-YN viruses and reduced syncytial formation by these viruses. The enzyme had little effect on replication and cytopathic effect of LT-910N and LT-YN viruses. The reason for these effects of the exogenous neuraminidase is discussed.
(Keyword)
Animals / Cell Line / Culture Media / Dogs / Genetic Variation / Neuraminidase / Parainfluenza Virus 3, Human / Respirovirus / Viral Plaque Assay / Viral Proteins
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 6271683
A. Ishimoto, Akio Adachi, K. Sakai, T. Yorifuji and S. Tsuruta : Rapid emergence of mink cell focus-forming (MCF) virus in various mice infected with NB-tropic Friend virus, Journal of Virology, Vol.113, No.24, 644-655, 1981.
233.
Akio Adachi, Tadahito Kanda and Hiroshi Shibuta : Isolation and characterization of temperature-sensitive mutants of Sendai virus, Microbiology and Immunology, Vol.24, No.11, 1053-1068, 1980.
(Summary)
Sixteen temperature-sensitive mutants of Sendai virus were isolated from mutagenized stocks (10 mutants, designated numerically) and persistently infected cultures (6 mutants, designated alphabetically). Based on complementation tests, virion-associated activities, thermal inactivation, and viral RNA and hemadsorbing antigen synthesis as well as virion production in chick lung embryo cells at nonpermissive temperature, these mutants were divided into seven groups as follows. i) HANA group mutants (ts-5, -9, -10, -201), defective in hemagglutinin-neuraminidase protein, complementation group I. ii) F group mutants (ts-18, -108), defective in hemolytic and cell-fusing activity, complementation group II. iii) Ts-43, defective in RNA polymerase activity, complementation group III. iv) Ts-23, defective in RNA polymerase activity, interfered with the other mutants in complementation tests. v) Ts-25, defective in the incorporation of hemagglutinin-neuraminidase protein into the virion at the stage of virus assembly. vi) Ts-110, belongs to F group mutants on one hand, but is considered to carry another undetermined defect. vii) C group (carrier culture-borne group) mutants (ts-a, -b, -c, -d, -e, -f), defective lesion not yet determined and belong to neither complementation group I nor II. Assignment of mutants in groups iv), v), vi), and vii) to complementation groups could not be achieved.
(Keyword)
Cell Line / Genetic Complementation Test / Hot Temperature / Lung / Mutation / Parainfluenza Virus 1, Human / Phenotype / RNA, Viral / Virus Cultivation
Hiroshi Shibuta, Tadahito Kanda, Akio Adachi and Yoshiyuki Yogo : Characterization of bovine parainfluenza virus type 3, Microbiology and Immunology, Vol.23, No.7, 617-628, 1979.
(Summary)
Bovine parainfluenza virus type 3 (PIV-3) has a buoyant density of 1.197. The RNA of PIV-3, like that of Sendai virus, is a single continuous chain which lacks polyadenylic acid sequences and tends to self-anneal to a marked extent. It has a sedimentation coefficient of 42S and a molecular weight of 4.5 X 10(6), being slightly smaller than Sendai virus RNA (47S, 5.3 X 10(6)). PIV-3 has 5 main structural proteins, of which 2 are glycoproteins. The molecular weights of protein 1, protein 2, protein 3, glycoprotein 1, and glycoprotein 2 were estimated to be 79,000, 68,000, 35,000, 69,000, and 55,000, respectively. Protein 2 was suggested to be nucleocapsid protein.
Hiroshi Shibuta, Akio Adachi, Tadahito Kanda and Hiroyuki Shimada : Experimental parainfluenzavirus infection. 1. Hydrocephalus of mice due to infection with parainfluenza virus type 1 and type 3, Microbiology and Immunology, Vol.22, No.8, 505-508, 1978.
M Kurokawa, Hajime A Koyama, S Yasuoka and Akio Adachi : Influenza virus overcomes apoptosis by rapid multiplication, International Journal of Molecular Medicine, Vol.3, No.5, 527-530, 1999.
(Summary)
The kinetics of apoptotic fragmentation of the chromosomal DNA was determined in the influenza virus-infected MDCK, HeLa and KB cells, respectively. Comparison of these kinetics with the kinetics of virus multiplication revealed that the multiplication of influenza virus was observed only when apoptosis was induced after the production of progeny virus in the infected cells. The extent of apoptotic response was reversely correlated with the permissiveness of the cells.
(Keyword)
Animals / apoptosis / Cell Line / DNA Fragmentation / Dogs / Humans / Influenza A virus / Kinetics / Virulence / Virus Replication
Hajime A Koyama, H Irie, Tomoharu Fukumori, S Hata, S Iida, H Akari and Akio Adachi : Role of virus-induced apoptosis in a host defense mechanism against virus infection, The Journal of Medical Investigation : JMI, Vol.45, No.1-4, 37-45, 1998.
(Summary)
Many animal viruses are known to induce apoptosis in infected cells. This virus-induced apoptosis has been often described as a mechanism of host defense against virus infection, based on the finding that mutants of an insect virus with the ability to induce extensive apoptosis in some cells cannot grow in the same cells. In animal virus infection, we have shown that (1) viruses can somehow overcome this defense mechanism and that (2) virus multiplication in the apoptotic cells is not as completely suppressed as in the insect virus infection. These results suggest that, in the case of animal viruses, the virus-induced apoptosis does not play the same role in the host defense system as in insect cells. However, by examining the virus infection under the conditions comparable to the infection in vivo, we demonstrated the defensive role of apoptosis in animal virus infection.
(Tokushima University Institutional Repository: 111615, PubMed: 9864963)
3.
Hirofumi Akari, Akio Adachi, Ki-Hoan Nam, K Mori, Isao Otani, Keiji Terao and Yasuhiro Yoshikawa : Early depletion of peripheral blood CD4+CD8+ T lymphocytes in cynomolgus macaques by SIVmac infection: implication of Nef, 12th World AIDS Conference, Vol.1, 69-73, 1998.
4.
Y Kubo, K Higo, H Kobayashi, T Ono, Y Iwama, S Yanagawa, Akio Adachi and A Ishimoto : Possible origin of murine AIDS-inducing sequence, Leukemia, Vol.11, No.Suppl 3, 163-166, 1997.
5.
Akio Adachi, Meiko Kawamura, Kenzo Tokunaga and Hiroyuki Sakai : Methods for HIV/SIV gene analysis, Viral Genome Methods, 45-53, 1996.
6.
R Shibata and Akio Adachi : SIV/HIV recombinants and their use in studying biological properties, AIDS Research and Human Retroviruses, Vol.8, No.3, 403-409, 1992.
(Summary)
A series of chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency viruses (SIV) were constructed. Viability of the recombinant viruses was dependent on the position of recombination. Infectious chimeric viruses between HIV-1 and SIVAGM (isolated from an African green monkey) and those between HIV-1 and SIVMAC (isolated from a rhesus monkey) were examined for host cell tropism. Viral determinants that restrict the replication of SIVAGM in human MT-4 cells and that of HIV-1 in macaque monkey peripheral blood mononuclear cells (PBMC) mapped to the 5' half of the virus genome. One HIV-1/SIVMAC chimera which contained the HIV-1 env gene was shown to replicate in macaque PBMC in vitro and to infect macaque monkeys in vivo. This HIV-1/SIVMAC chimera will be useful for a variety of AIDS pathogenesis and vaccine studies.
R Shibata, Akio Adachi, H Sakai, A Ishimoto, T Miura and M Hayami : Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2, Journal of Medical Primatology, Vol.19, No.3-4, 217-225, 1990.
(Summary)
We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus.
Takaaki Koma, Shun Adachi, Naoya Doi, Akio Adachi and Masako Nomaguchi : Toward Understanding Molecular Bases for Biological Diversification of Human Coronaviruses: Present Status and Future Perspectives., Frontiers in Microbiology, Vol.11, Aug. 2020.
(Summary)
replication for various accessory proteins encoded by the variable 3' one-third portion of the CoV genome mostly remain to be determined. Importantly, the genomic sequences/structures closely linked to the high CoV recombination are poorly investigated and elucidated. Also, determinants for adaptation and pathogenicity have not been systematically investigated. We summarize here these research situations. Among conceivable projects, we are especially interested in the underlying molecular mechanism by which the observed CoV diversification is generated. Finally, as virologists, we discuss how we handle the present difficulties and propose possible research directions in the medium or long term.
Masako Nomaguchi, Naoya Doi, Takaaki Koma and Akio Adachi : HIV-1 mutates to adapt in fluxing environments., Microbes and Infection, Oct. 2018.
(Summary)
Human immunodeficiency virus type 1 (HIV-1) is specifically adapted for replication, persistence, transmission, and survival in humans. HIV-1 is highly mutable in nature, and well responds to a variety of environmental pressures by altering its genome sequences. In this review, we have described experimental evidence that demonstrates this phantasmagoric property of HIV-1.
T. Sultana, E.E. Nakayama, S. Tobita, M. Yokoyama, Y. Seki, A. Saito, Masako Nomaguchi, Akio Adachi, H. Akari, H. Sato and T. Shioda : Novel mutant human immunodeficiency virus type 1 strains with high degree of resistance to cynomolgus macaque TRIMCyp generated by random mutagenesis., The 23rd Conference on Retroviruses and Opportunistic Infections, Boston, USA, Feb. 2016.
2.
A Sato, Masako Nomaguchi, K Kono, E.E. Nakayama, T Shioda, T Yoshida, Y Yasutomi, T Matano, Akio Adachi and H Akari : Genotypic variation of cynomolgus monkey trim5alpha determines the susceptibility to monkey-tropic HIV-1 infection., XV International Congress of Virology, Sep. 2011.
3.
N Takahashi, A Saito, Masako Nomaguchi, Akio Adachi, H Akari and T Matano : Viral recovery from cynomolgus macaques controlling a simian-tropic HIV-1 challenge., XV International Congress of Virology, Sep. 2011.
4.
Ariko Miyake, Doi Naoya, Fujiwara Sachi, Akio Adachi and Masako Nomaguchi : Analysis of growth adaptive mutations in HIV-1 genome identifies a pol-intergrase region that enhances virion production in a cell-independent and codon triplet-dependent manner., The 10th Awaji International Forum on Infection and Immunity, Awaji, Japan, Sep. 2010.
5.
Mikako Fujita, Boonruang Khamsri, Fumiko Murao, Akiko Yoshida, Akiko Sakurai, Tsuneo Uchiyama, K Kamada, H Shirai, Yo Matsuo and Akio Adachi : Comparative study on the structure and cytopathogenic activity of HIV Vpr/Vpx proteins, 5th Awaji International Forum on Infection and Immunity, Sumoto, Japan, Sep. 2005.
Bao Quoc Le, Yokoyama Masaru, Naoya Doi, ICHINOMIYA Takumi, KOMODA Nanako, Tomoyuki Kondo, Akio Adachi, Kotani Osamu, Sato Hironori, Masako Nomaguchi and Takaaki Koma : The importance of a novel ITI triplet motif within Env V3 domain for R5-tropic HIV-1 replication, 第70回日本ウイルス学会学術集会, Sep. 2023.
3.
Takaaki Koma, RE Kuokku Bao, Naoya Doi, KOMODA Nanako, ICHINOMIYA Takumi, Tomoyuki Kondo, Akio Adachi and Masako Nomaguchi : Implications of Gag-NC and gRNA interactions in HIV-1 assembly, 第70回日本ウイルス学会学術集会, Sep. 2023.
4.
Tomoyuki Kondo, Takaaki Koma, Naoya Doi, RE Kuokku Bao, KOMODA Nanako, ICHINOMIYA Takumi, Akio Adachi and Masako Nomaguchi : Effect of naturally-occurring synonymous single-nucleotide mutations within the vpr-coding sequence on HIV-1 replication, 第70回日本ウイルス学会学術集会, Sep. 2023.
5.
Naoya Doi, Takaaki Koma, LE Quoc Bao, KOMODA Nanako, ICHINOMIYA Takumi, Tomoyuki Kondo, Akio Adachi and Masako Nomaguchi : PIM kinases suppress HIV virion production by inhibiting viral protein expression, 第70回日本ウイルス学会学術集会, Sep. 2023.
Tomoyuki Kondo, Takaaki Koma, Akio Adachi, Masako Nomaguchi and Naoya Doi : PIMキナーゼによるHIV型特異的な遺伝子発現調節の解析, 第36回日本エイズ学会学術集会・総会, Nov. 2022.
8.
Naoya Doi, Takaaki Koma, Chisato Gotohda, NAGASAKA Mari, Tomoyuki Kondo, Akio Adachi and Masako Nomaguchi : Importance of vpr-coding sequence in HIV-1 gene expression, 第69回日本ウイルス学会学術集会, Nov. 2022.
9.
Tomoyuki Kondo, Takaaki Koma, UDAGAWA Mei, OKUMURA Nozomi, Akio Adachi, Masako Nomaguchi and Naoya Doi : Analysis of HIV type-specific regulation of viral gene expression by PIM, 第69回日本ウイルス学会学術集会, Nov. 2022.
Takaaki Koma, Naoya Doi, Satoshi Nakashima, Akio Adachi and Masako Nomaguchi : Exploration for assembly-promoting factors involved in the early stage of HIV-1 Gag assembly, 第67回日本ウイルス学会学術集会, Oct. 2019.
17.
Masako Nomaguchi, Takaaki Koma, Naoya Doi, Hideki Yamamoto, Kyosuke Watanabe, Mai Takemoto and Akio Adachi : Molecular bases for determination of the Vif expression level by single-nucleotide mutations within SA1D2prox and by adaptive mutations in the HIV-1 genome, 第67回日本ウイルス学会学術集会, Oct. 2019.
18.
Naoya Doi, Takaaki Koma, Akio Adachi and Masako Nomaguchi : Analysis of molecular mechanism on species-specific growth enhancement by a single-amino acid mutation in V3 tip of CCR5-tropic HIV-1 Env, 第67回日本ウイルス学会学術集会, Oct. 2019.
Shoko Nakanishi, Sakimi Watanabe, Naoya Doi, Takaaki Koma, Akio Adachi and Masako Nomaguchi : Single-amino acid mutations in V1 and C4 domains of HIV-1 Env cooperatively enhance viral replication ability by increasing CD4 affinity, 第65回日本ウイルス学会学術集会, Oct. 2017.
25.
Sakimi Watanabe, Shoko Nakanishi, Takaaki Koma, Naoya Doi, Akio Adachi and Masako Nomaguchi : Low vif type of HIV-1 SA1D2prox variants can adaptively mutate to enhance the Vif expression under the strong restrictive condition, 第65回日本ウイルス学会学術集会, Oct. 2017.
26.
Naoya Doi, Takaaki Koma, Shoko Nakanishi, Sakimi Watanabe, Masako Nomaguchi and Akio Adachi : Generation of R5-tropic HIV-1rmt clones with an enhanced replication ability by adaptive Env domain swapping, 第65回日本ウイルス学会学術集会, Oct. 2017.
27.
Akio Adachi, Naoya Doi, Takaaki Koma, Shoko Nakanishi, Sakimi Watanabe and Masako Nomaguchi : Linkage analysis of HIV-1 vif production level and SLSA1 structure/energy stability, 第65回日本ウイルス学会学術集会, Oct. 2017.
28.
Takaaki Koma, Naoya Doi, 宮川 敬, 梁 明秀, Akio Adachi and Masako Nomaguchi : Role for amino acid residues S149 and I150 of the Gag-CA linker domain in the late HIV-1 replication phase, 第65回日本ウイルス学会学術集会, Oct. 2017.
29.
酒井 遥介, 藤本 薫平, 土肥 直哉, Masako Nomaguchi and Akio Adachi : Studies on the adaptation process of HIV-1 Env in macaque cells, 第64回日本ウイルス学会学術集会, Oct. 2016.
30.
A Kawakami, A Himeno, M Kikukawa, Y Ishida, Masako Nomaguchi, Akio Adachi and T Miura : Generation of neutralization-resistant and CCR5 tropic HIV-1rmt, 第64回日本ウイルス学会学術集会, Oct. 2016.
31.
N Doi, Chieko Ishifune, Koji Yasutomo, T Miura, Y Sakai, K Fujimoto, S Harada, K Yoshimura, Masako Nomaguchi and Akio Adachi : Replication and pathogenicity of HIV-1rmt: towards evaluation of viral growth ability in gut-derived cells, 第64回日本ウイルス学会学術集会, Oct. 2016.
32.
Masako Nomaguchi, 土肥 直哉, 藤本 薫平, 酒井 遥介, 中西 祥子 and Akio Adachi : Identification of cis-elements involved in the HIV-1 vif mRNA production, 第64回日本ウイルス学会学術集会, Oct. 2016.
33.
Akio Adachi, 土肥 直哉, 酒井 遥介, 藤本 薫平 and Masako Nomaguchi : An ultra-low vif type of HIV-1 SA1D2prox variant can adapt and evolve under the high level of APOBEC3G, 第64回日本ウイルス学会学術集会, Oct. 2016.
34.
藤本 薫平, 土肥 直哉, 酒井 遥介, Akio Adachi and Masako Nomaguchi : Effects of mutations in HIV-1 Gag-CA helix 7 and linker domain on the virion production, 第64回日本ウイルス学会学術集会, Oct. 2016.
35.
S. Tahmina, E.E. Nakayama, S. Tobita, A. Saito, Masako Nomaguchi, Akio Adachi, H. Akari and T. Shioda : Novel mutant HIV-1 strains with high degree of resistance to cynomolgus macaque TRIMCyp generated by random mutagenesis., The 63rd Annual Meeting of the Japanese Society for Virology, Nov. 2015.
Analysis of molecular basis for modulation of HIV-1 replication by naturally-occurring single nucleotide polymorphisms within the SLSA1 structure of the pol gene (Project/Area Number: 26460556 )
Basic study on HIV-1 replication and pathogenicity in host individuals: functional analysis of accessory proteins (Project/Area Number: 26293104 )
New anti-HIV/SIV cellular-factors counteracted by Vpx: their search and identification (Project/Area Number: 24659208 )