Takaaki Koma, Naoya Doi, Le Quoc Bao, Tomoyuki Kondo, Akio Adachi and Masako Nomaguchi : HIV-1 Replication and Pathogenicity: Lessons from Macaque-Tropic HIV-1 Derivatives, IntechOpen, London, Sep. 2023.
2.
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.
Takeo Minamikawa, Takaaki Koma, Akihiro Suzuki, Takahiko Mizuno, Kentaro Nagamatsu, Hideki Arimochi, Koichiro Tsuchiya, Kaoru Matsuoka, Takeshi Yasui, Koji Yasutomo and Masako Nomaguchi : Quantitative evaluation of SARS-CoV-2 inactivation using a deep ultraviolet light-emitting diode., Scientific Reports, Vol.11, 5070, 2021.
(Summary)
for 300 nm are required to inactivate 99.9% of SARS-CoV-2. Our results provide quantitative antiviral effects of DUV irradiation on SARS-CoV-2, serving as basic knowledge of inactivation technologies against SARS-CoV-2.
Yoshihiko Ueno, Yosuke Seki, Masashi Akaike and Masako Nomaguchi : 教育連動型AO入試の設計と実施ーー地方国立大学における研究医の養成・確保をめざしてーー, The Journal of University Admissions Research, No.30, 207-213, 2020.
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.
K Miyakawa, S Matsunaga, M Yokoyama, Masako Nomaguchi, Y Kimura, M Nishi, H Kimura, H Sato, H Hirano, T Tamura, H Akari, T Miura, A Adachi, T Sawasaki, N Yamamoto and A Ryo : PIM kinases facilitate lentiviral evasion from SAMHD1 restriction via Vpx phosphorylation., Nature Communications, Vol.10, No.1, 1844, 2019.
(Summary)
Lentiviruses have evolved to acquire an auxiliary protein Vpx to counteract the intrinsic host restriction factor SAMHD1. Although Vpx is phosphorylated, it remains unclear whether such phosphorylation indeed regulates its activity toward SAMHD1. Here we identify the PIM family of serine/threonine protein kinases as the factors responsible for the phosphorylation of Vpx and the promotion of Vpx-mediated SAMHD1 counteraction. Integrated proteomics and subsequent functional analysis reveal that PIM family kinases, PIM1 and PIM3, phosphorylate HIV-2 Vpx at Ser13 and stabilize the interaction of Vpx with SAMHD1 thereby promoting ubiquitin-mediated proteolysis of SAMHD1. Inhibition of the PIM kinases promotes the antiviral activity of SAMHD1, ultimately reducing viral replication. Our results highlight a new mode of virus-host cell interaction in which host PIM kinases facilitate promotion of viral infectivity by counteracting the host antiviral system, and suggest a novel therapeutic strategy involving restoration of SAMHD1-mediated antiviral response.
(Keyword)
Biphenyl Compounds / Cell Line, Tumor / HEK293 Cells / HIV Infections / HIV-2 / Host-Pathogen Interactions / Humans / Imidazoles / Immune Tolerance / Molecular Dynamics Simulation / Monocytes / Phosphorylation / Protein Binding / Protein Kinase Inhibitors / Protein Processing, Post-Translational / Protein-Serine-Threonine Kinases / Proteolysis / Proteomics / Proto-Oncogene Proteins / Proto-Oncogene Proteins c-pim-1 / Pyridazines / Recombinant Proteins / SAM Domain and HD Domain-Containing Protein 1 / Serine / Thiazolidines / Viral Regulatory and Accessory Proteins / Virus 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.
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.
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.
Li Yu, Masako Nomaguchi, R Padmanabhan and Lewis Markoff : Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication., Virology, Vol.374, No.1, 170-185, 2008.
(Summary)
We initially studied requirements for 5' and 3' terminal regions (TRs) in flavivirus negative strand synthesis in vitro. Purified West Nile (WNV) and dengue-2 (DV2) RNA polymerases were both active with all-WNV or all-DV2 subgenomic RNAs containing the 5'- and 3'TRs of the respective genomes. However, subgenomic RNAs in which the 5'-noncoding region (5'NCR) or the 5'ORF (nts 100-230) in the 5'TR were substituted by analogous sequences derived from the heterologous genome were modestly to severely defective as templates for either polymerase. We also evaluated the infectivity of substitution mutant WNV genome-length RNAs. All WNV RNAs containing the DV2 3'SL were unable to replicate. However, WNV RNAs containing substitutions of the 5'NCR, the capsid gene, and/or 3'NCR nt sequences upstream from the WNV 3'SL, by the analogous DV2 nt sequences, were infectious. Combined results suggested that replication was not dependent upon species homology between the 3'SL and NS5.
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.
Akihide Tanimoto, Ke-Yong Wang, Yoshitaka Murata, Satoshi Kimura, Masako Nomaguchi, Sei Nakata, Masato Tsutsui and Yasuyuki Sasaguri : Histamine upregulates the expression of inducible nitric oxide synthase in human intimal smooth muscle cells via histamine H1 receptor and NF-κB signaling pathway., Arteriosclerosis, Thrombosis, and Vascular Biology, Vol.27, No.7, 1556-1561, 2007.
(Summary)
Histamine stimulates intimal SMCs to increase iNOS expression via H1 receptors and NF-κB signaling pathway. Histamine could be one of NO-regulating factors, by inducing iNOS expression in intimal SMCs, and may be related to atherogenesis.
(Keyword)
Analysis of Variance / Blotting, Northern / Blotting, Western / Cells, Cultured / Gene Expression Regulation / Histamine / Humans / Muscle, Smooth, Vascular / NF-κB / Nitric Oxide Synthase Type II / Probability / Receptors, Histamine H1 / Sensitivity and Specificity / Signal Transduction / Tunica Intima / Up-Regulation
Masako Nomaguchi, Tadahisa Teramoto, Li Yu, Lewis Markoff and R Padmanabhan : Requirements for West Nile virus (-)- and (+)-strand subgenomic RNA synthesis in vitro by the viral RNA-dependent RNA polymerase expressed in Escherichia coli., The Journal of Biological Chemistry, Vol.279, No.13, 12141-12151, 2003.
(Summary)
RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (-)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (-)- and (+)-strand template RNAs containing 5'- and 3'-terminal regions (TR) with the conserved sequence elements. Our results show that both 5'- and 3'-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3'-TR of the (-)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5'-CYC motif is critical for (-)-strand RNA synthesis but neither the (-)-strand 5'- nor 3'-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5'-cap inhibits the (-)-strand RNA synthesis from the 3' fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that "cyclization" of the viral RNA play a role for (-)-strand RNA synthesis but not for (+)-strand RNA synthesis.
Masako Nomaguchi, Matt Ackermann, Changsuek Yon, Shihyun You, R Padmanabhan and R Padmanbhan : De novo synthesis of negative-strand RNA by Dengue virus RNA-dependent RNA polymerase in vitro: nucleotide, primer, and template parameters., Journal of Virology, Vol.77, No.16, 8831-8842, 2003.
(Summary)
By using a purified dengue virus RNA-dependent RNA polymerase and a subgenomic 770-nucleotide RNA template, it was shown previously that the ratio of the de novo synthesis product to hairpin product formed was inversely proportional to increments of assay temperatures (20 to 40 degrees C). In this study, the components of the de novo preinitiation complex are defined as ATP, a high concentration of GTP (500 micro M), the polymerase, and the template RNA. Even when the 3'-terminal sequence of template RNA was mutated from -GGUUCU-3' to -GGUUUU-3', a high GTP concentration was required for de novo initiation, suggesting that high GTP concentration plays a conformational role. Furthermore, utilization of synthetic primers by the polymerase indicated that AGAA is the optimal primer whereas AG, AGA, and AGAACC were inefficient primers. Moreover, mutational analysis of the highly conserved 3'-terminal dinucleotide CU of the template RNA indicated that change of the 3'-terminal nucleotide from U to C reduced the efficiency about fivefold. The order of preference for the 3'-terminal nucleotide, from highest to lowest, is U, A - G, and C. However, change of the penultimate nucleotide from C to U did not affect the template activity. A model consistent with these results is that the active site of the polymerase switches from a "closed" form, catalyzing de novo initiation through synthesis of short primers, to an "open" form for elongation of a double-stranded template-primer.
(Keyword)
Base Sequence / DNA Primers / Dengue Virus / RNA Replicase / RNA, Viral / Templates, Genetic
A. Tanimoto, Y. Murata, Masako Nomaguchi, A. Kimura, N. Arima, H. Xu, T. Hamada and Y. Sasaguri : Histamine increases the expression of LOX-1 via H2 receptor in human monocytic THP-1 cells, FEBS Letters, Vol.508, No.3, 345-349, 2001.
(Summary)
Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a member of the scavenger receptor family, and is known to be expressed in monocytes/macrophages. We investigated the effect of histamine on the expression of LOX-1 in cells of the human monocytic leukemia cell line THP-1. Histamine as well as forskolin and dibutyryl cyclic AMP (Bt2-cAMP) stimulated the THP-1 monocytes to express the LOX-1 gene at the transcription level. This histamine effect on LOX-1 gene expression, via the histamine H2 receptor-mediated cAMP signal transduction pathway, was reduced after differentiation of the cells into macrophages, even though forskolin and Bt2-cAMP still enhanced the gene expression. The alteration of the responsiveness of LOX-1 expression to histamine was related to suppressed expression of the H2 receptor in THP-1 macrophages. The switch of the predominant class of histamine receptors between H1 and H2 would modulate the effects of histamine on LOX-1 gene expression in monocytes and macrophages, and therefore, would play a certain role in the inflammatory aspects of atherogenesis.
Hiroyuki Nonogaki, Masako Nomaguchi, Yukio Morohashi and Hisashi Matsushima : Development and localization of endo-beta-mannanase in the embryo of germinating tomato seeds, Journal of Experimental Botany, Vol.49, 1501-1507, 1998.
H. Nonogaki, Masako Nomaguchi, H. Okumoto, Y. Kaneko, H. Matsushima and Y. Morohashi : Temporal and spatial pattern of the biochemical activation of the endosperm during and following imbibition of tomato seeds, Plant, Vol.102, 236-242, 1998.
75.
H. Nonogaki, Masako Nomaguchi and Y. Morohashi : Endo-beta-mannanase in the ecdosperm of germinated tomato seeds, Physiol. Plant., Vol.94, 328-334, 1995.
76.
Masako Nomaguchi, H. Nonogaki and Y. Morohashi : Development of galactomannan-hydrolyzing activity in the micropylar endosperm tip of tomato seed prior to germination, Physiol. Plant., Vol.94, 105-109, 1995.
Review, Commentary:
1.
Takeshi Yasui, Takeo Minamikawa, Yu Tokizane, Naoya Kuse, Takaaki Koma, Takao Ueda and Masako Nomaguchi : 目に見えない光が切り拓く『光の世紀』, Journal of the Japan Society for Precision Engineering, Vol.89, No.8, 587-591, Aug. 2023.
Takeo Minamikawa, Takaaki Koma, Suzuki Akihiro, Kentaro Nagamatsu, Takeshi Yasui, Koji Yasutomo and Masako Nomaguchi : Inactivation of SARS-CoV-2 by deep ultraviolet light emitting diode: A review, Japanese Journal of Applied Physics, Vol.60, No.9, 090501, Aug. 2021.
Takeo Minamikawa, Takaaki Koma, 鈴木 昭浩, Kentaro Nagamatsu, Takeshi Yasui, Koji Yasutomo and Masako Nomaguchi : 深紫外LEDによる新型コロナウイルス不活化への試み, OPTRONICS, Vol.40, No.6, 132-137, May 2021.
5.
Takeo Minamikawa, Takaaki Koma, 鈴木 昭浩, Kentaro Nagamatsu, Takeshi Yasui, Koji Yasutomo and Masako Nomaguchi : 深紫外LEDを用いた新型コロナウイルスの不活化, O plus E, Vol.43, No.2, 137-142, Mar. 2021.
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.
Noriaki Minakawa, Noriko Saito-Tarashima, Takaaki Koma, NAOTO Hinotani, YOSHIDA Keigo, OGASA Moka, AKIHO Murai, INOUE Shuya, Tomoyuki Kondo, Naoya Doi, Koichi Tsuneyama and Masako Nomaguchi : 3-Deazaguanosine exhibits anti-SARS-CoV-2 activity and blocks the development of COVID-19 pneumonis in hamsters., Supra FIBER International Summit for Nucleic Acids (S-FISNA) 2024, Mar. 2024.
2.
Nogi Yuhei, Noriko Saito-Tarashima, Takaaki Koma, Masako Nomaguchi and Noriaki Minakawa : Development of the 4'-thiomodified siRNAs against SARS-CoV-2, 14th AFMC International Medicinal Chemistry Symposium, Jun. 2023.
3.
Naruki Miu, Saito Motofumi, Tomita Masaru, Masako Nomaguchi and Kanai Akio : Computational analysis of the acquisition and evolution of the vpu gene in Human Immunodeficiency Virus-1, The 28th Annual Meeting of the RNA Society, Singapore, May 2023.
4.
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.
5.
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.
6.
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.
7.
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.
Proceeding of Domestic Conference:
1.
YOSHIDA Keigo, NAOTO Hinotani, OGASA Moka, Noriko Saito-Tarashima, Takaaki Koma, Masako Nomaguchi and Noriaki Minakawa : SARS-CoV-2活性を発揮する3-デアザグアノシンの発見と作用メカニズム解明, 日本薬学会第144年会, Mar. 2024.
2.
YUHEI Nogi, Noriko Saito-Tarashima, Takaaki Koma, Jun Tsukimoto, Masako Nomaguchi and Noriaki Minakawa : 抗SARS-CoV-2活性を指標とした4'-チオ修飾siRNAの最適化, 日本薬学会第144年会, Mar. 2024.
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.
7.
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.
8.
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.
9.
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.
14.
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.
15.
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.
Tomoyuki Kondo, Takaaki Koma, Akio Adachi, Masako Nomaguchi and Naoya Doi : PIMキナーゼ及びPIM阻害剤によるHIV種特異的な遺伝子発現と複製への影響, 第265回徳島医学会学術集会, Jul. 2022.
18.
Naruki Miu, Saito Motofumi, Tomita Masaru, Masako Nomaguchi and Kanai Akio : The acquisition and molecular evolution of the vpu gene in HIV-1, 第22回日本RNA学会, Jul. 2022.
19.
Miu Naruki, Motofumi Saito, Masaru Tomita, Masako Nomaguchi and Akio Kanai : The acquisition and molecular evolution of the vpu gene in HIV-1, 第23回日本RNA学会, Jul. 2022.
Naruki Miu, Saito Motofumi, Tomita Masaru, Masako Nomaguchi and Kanai Akio : Detailed classification of Vpu proteins and the possible acquisition of the gene in Human immunodeficiency Virus-1, 第44回 日本分子生物学会年会, Dec. 2021.
22.
Takaaki Koma, Mitsuki Ishizue, Chizuko Tsukada, Chiaki Tokaji, Akio Adachi, Masako Nomaguchi and Naoya Doi : PIMキナーゼ阻害剤がHIV種特異的に複製に及ぼす影響の解析(若手優秀演題), 第35回日本エイズ学会学術集会・総会, Nov. 2021.
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.
31.
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.
32.
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.
41.
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.
42.
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.
43.
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.
44.
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.
45.
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.
46.
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.
47.
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.
48.
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.
49.
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.
50.
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.
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.
Treatment and prevention of RNA virus infections based on 4'-thionucleic acid technology (Project/Area Number: 21H02606 )
Elucidation of the regulatory basis for alternative splicing towards controlling HIV-1 replication (Project/Area Number: 17K08860 )
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 )