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.
(要約)
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.
(要約)
Understanding molecular bases for viral entry into cells leads to the elucidation of one of major viral survival strategies, and thus to the development of new effective antiviral measures. As experimentally shown recently, HIV-1 is highly mutable and adaptable in growth-restrictive cells such as those of macaque origin. HIV-1 initiates its infection by sequential interactions of Env-gp120 with two cell surface receptors, CD4 and CCR5. A recent epoch-making structural study has disclosed that CD4-induced conformation of gp120 is stabilized upon binding of CCR5 to the CD4-gp120 complex, whereas its biological significance remains totally unknown. Here, from a series of mutations found in our extensive studies, we identified a single-amino acid adaptive mutation at the V3 loop tip of Env-gp120 critical for its interaction with both CD4 and CCR5 in a host cell species-specific way. This remarkable finding would certainly provoke and accelerate studies to precisely clarify the HIV-1 entry mechanism.
Naoya Doi, Takaaki Koma, Akio Adachi and Masako Nomaguchi : Expression Level of HIV-1 Vif Can Be Fluctuated by Natural Nucleotide Variations in the vif-Coding and Regulatory SA1D2prox Sequences of the Proviral Genome., Frontiers in Microbiology, Vol.10, 2019.
Naoya Doi, Takaaki Koma, Akio Adachi and Masako Nomaguchi : Role for Gag-CA Interdomain Linker in Primate Lentiviral Replication., Frontiers in Microbiology, Vol.10, 2019.
(要約)
-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.
(要約)
HIV-1 particle production and infection are highly ordered processes. Viral Gag proteins play a central role in the assembly and disassembly of viral molecules. Of these, capsid protein (CA) is a major contributor to the Gag-Gag interactions. CA consists of two structured domains, i.e., N-terminal (NTD) and C-terminal (CTD) domains, connected by an unstructured domain named interdomain linker. While multiple regions in the NTD and CTD domains are reported to play roles in virion morphogenesis and infectivity, the roles of the linker region in Gag assembly and virus particle formation remain elusive. In this report, we show by biological and molecular analyses that the linker region functions as an intramolecular modulator to tune Gag assembly, virion production, and viral infectivity. Our study thus illustrates a hitherto unrecognized mechanism, an allosteric regulation of CA structure by the disordered protein element, for HIV-1 replication.
Naoya Doi, Masaru Yokoyama, Takaaki Koma, Osamu Kotani, Hironori Sato, Akio Adachi and Masako Nomaguchi : Concomitant Enhancement of HIV-1 Replication Potential and Neutralization-Resistance in Concert With Three Adaptive Mutations in Env V1/C2/C4 Domains., Frontiers in Microbiology, Vol.10, 2019.
(要約)
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.
(要約)
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.
(要約)
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.
(要約)
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.
(要約)
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.
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.
(要約)
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.
(要約)
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.