Involvement of a Rarely Used Splicing SD2b Site in the Regulation of HIV-1 vif mRNA Production as Revealed by a Growth-Adaptive Mutation.

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 vif 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 vif mRNA production through increasing the splicing site usage of SA1. Our results here revealed the importance of the SD2b nucleotide sequence in producing vif mRNA involved in the HIV-1 adaptation and of mutual antagonism between Vif and APOBEC3 proteins in HIV-1 adaptation/evolution and survival.

Special Issue "Cell, Organoid and Animal Models to Study Pathogenic Human RNA Viruses".

Numerous species of RNA viruses pathogenic for humans are present worldwide [...].

The Expression Level of HIV-1 Vif Is Optimized by Nucleotide Changes in the Genomic SA1D2prox Region during the Viral Adaptation Process.

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.

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.

Molecular interactions of the variable envelope gp120 subunit of HIV-1 with two cellular receptors are the first step of viral infection, thereby playing pivotal roles in determining viral infectivity and cell tropism. However, the underlying regulatory mechanisms for interactions under gp120 spontaneous variations largely remain unknown. Here, we show an allosteric mechanism in which a single gp120 mutation remotely controls the ternary interactions between gp120 and its receptors for the switch of viral cell tropism. Virological analyses showed that a G310R substitution at the tip of the gp120 V3 loop selectively abolished the viral replication ability in human cells, despite evoking enhancement of viral replication in macaque cells. Molecular dynamics (MD) simulations predicted that the G310R substitution at a site away from the CD4 interaction site selectively impeded the binding ability of gp120 to human CD4. Consistently, virions with the G310R substitution exhibited a reduced binding ability to human lymphocyte cells. Furthermore, the G310R substitution influenced the gp120-CCR5 interaction in a CCR5-type dependent manner as assessed by MD simulations and an infectivity assay using exogenously expressed CCR5s. Interestingly, an I198M mutation in human CCR5 restored the infectivity of the G310R virus in human cells. Finally, MD simulation predicted amino acid interplays that physically connect the V3 loop and gp120 elements for the CD4 and CCR5 interactions. Collectively, these results suggest that the V3 loop tip is a cis-allosteric regulator that remotely controls intra- and intermolecular interactions of HIV-1 gp120 for balancing ternary interactions with CD4 and CCR5. IMPORTANCE Understanding the molecular bases for viral entry into cells will lead to the elucidation of one of the major viral survival strategies, and thus to the development of new effective antiviral measures. As 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 the biological significance of this 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 could certainly provoke and accelerate studies to precisely clarify the HIV-1 entry mechanism.

Toward Understanding Molecular Bases for Biological Diversification of Human Coronaviruses: Present Status and Future Perspectives.

Human coronaviruses (HCoVs) are of zoonotic origins, and seven distinct HCoVs are currently known to infect humans. While the four seasonal HCoVs appear to be mildly pathogenic and circulate among human populations, the other three designated SARS-CoV, MERS-CoV, and SARS-CoV-2 can cause severe diseases in some cases. The newly identified SARS-CoV-2, a causative virus of COVID-19 that can be deadly, is now spreading worldwide much more efficiently than the other two pathogenic viruses. Despite evident differences in these properties, all HCoVs commonly have an exceptionally large genomic RNA with a rather peculiar gene organization and have the potential to readily alter their biological properties. CoVs are characterized by their biological diversifications, high recombination, and efficient adaptive evolution. We are particularly concerned about the high replication and transmission nature of SARS-CoV-2, which may lead to the emergence of more transmissible and/or pathogenic viruses than ever before. Furthermore, novel variant viruses may appear at any time from the CoV pools actively circulating or persistently being maintained in the animal reservoirs, and from the CoVs in infected human individuals. In this review, we describe knowns of the CoVs and then mention their unknowns to clarify the major issues to be addressed. Genome organizations and sequences of numerous CoVs have been determined, and the viruses are presently classified into separate phylogenetic groups. Functional roles in the viral replication cycle in vitro of non-structural and structural proteins are also quite well understood or suggested. In contrast, those in the in vitro and in vivo 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.

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.

Vif is required for HIV-1 replication in natural target cells by counteracting host restriction factors, APOBEC3 (A3) proteins. We recently demonstrated that Vif expression level can be changed by naturally occurring single-nucleotide variations within SA1D2prox of the HIV-1 genome. We also found that levels for vif/vpr mRNAs are inversely correlated. While amino acid sequence per se is critical for functionality, Vif expression level modulated by signal sequences in its coding region is likely to be important as well. There are two splicing sites in the region involved in vpr expression. To reveal possible fluctuations of Vif-expression level, we examined SA1D2prox and vif gene by chimeric approaches using HIV-1 subtypes B and C with distinct anti-A3 activity. In this report, recombinant clones in subtype B backbone carrying chimeric sequences with respect to SA1D2prox/vif and those within the vif-coding region were generated. Of these, clones containing vif-coding sequence of subtype C, especially its 3' region, expressed vif/Vif at a decreased level but did at an increased level for vpr/Vpr. Clones with reduced vif/Vif level grew similarly or slightly better than a parental clone in weakly A3G-positive cells but more poorly in highly A3G-expressing cells. Three clones with this property were also tested for their A3-degrading activity. One of the clones appeared to have some defect in addition to the poor ability to express vif/Vif. Taken all together, our results show that natural variations in the SA1D2prox and vif-coding region can change the Vif-expression level and affect the HIV-1 replication potential.

Role for Gag-CA Interdomain Linker in Primate Lentiviral Replication.

Gag proteins underlie retroviral replication by fulfilling numerous functional roles at various stages during viral life cycle. Out of the four mature proteins, Gag-capsid (CA) is a major component of viral particles, and has been most well studied biogenetically, biochemically and structurally. Gag-CA is composed of two structured domains, and also of a short stretch of disordered and flexible interdomain linker. While the two domains, namely, N-terminal and C-terminal domains (NTD and CTD), have been the central target for Gag research, the linker region connecting the two has been poorly studied. We recently have performed systemic mutational analyses on the Gag-CA linker region of HIV-1 by various experimental and in silico systems. In total, we have demonstrated that the linker region acts as a cis-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.

Allosteric Regulation of HIV-1 Capsid Structure for Gag Assembly, Virion Production, and Viral Infectivity by a Disordered Interdomain Linker.

The retroviral Gag capsid (Gag-CA) interdomain linker is an unstructured peptide segment connecting structured N-terminal and C-terminal domains. Although the region is reported to play roles in virion morphogenesis and infectivity, underlying molecular mechanisms remain unexplored. To address this issue, we determined biological and molecular phenotypes of HIV-1 CA linker mutants by experimental and in silico approaches. Among the nine linker mutants tested, eight exhibited attenuation of viral particle production to various extents mostly in parallel with a reduction in viral infectivity. Sucrose density gradient, confocal microscopy, and live-cell protein interaction analyses indicated that the defect is accompanied by attenuation of Gag-Gag interactions following Gag plasma membrane targeting in the cells. In silico analyses revealed distinct distributions of interaction-prone hydrophobic patches between immature and mature CA proteins. Molecular dynamics simulations predicted that the linker mutations can allosterically alter structural fluctuations, including the interaction surfaces apart from the mutation sites in both the immature and mature CA proteins. These results suggest that the HIV-1 CA interdomain linker is a cis-modulator of the CA interaction surfaces to optimize efficiency of Gag assembly, virion production, and viral infectivity.IMPORTANCE 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 the interdomain linker. While multiple regions in the NTD and CTD 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 study, we showed 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.

PIM kinases facilitate lentiviral evasion from SAMHD1 restriction via Vpx phosphorylation.

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.

Concomitant Enhancement of HIV-1 Replication Potential and Neutralization-Resistance in Concert With Three Adaptive Mutations in Env V1/C2/C4 Domains.

HIV-1 Env protein functions in the entry process and is the target of neutralizing antibodies. Its intrinsically high mutation rate is certainly one of driving forces for persistence/survival in hosts. For optimal replication in various environments, HIV-1 Env must continue to adapt and evolve through balancing sometimes incompatible function, replication fitness, and neutralization sensitivity. We have previously reported that adapted viruses emerge in repeated and prolonged cultures of cells originally infected with a macaque-tropic HIV-1NL4-3 derivative. We have also shown that the adapted viral clones exhibit enhanced growth potentials both in macaque PBMCs and individuals, and that three single-amino acid mutations are present in their Env V1/C2/C4 domains. In this study, we investigated how lab-adapted and highly neutralization-sensitive HIV-1NL4-3 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.

CXCR4- and CCR5-Tropic HIV-1 Clones Are Both Tractable to Grow in Rhesus Macaques.

A major issue for present HIV-1 research is to establish model systems that reflect or mimic viral replication and pathogenesis actually observed in infected humans. To this end, various strategies using macaques as infection targets have long been pursued. In particular, experimental infections of rhesus macaques by HIV-1 derivatives have been believed to be best suited, if practicable, for studies on interaction of HIV-1 and humans under various circumstances. Recently, through in vitro 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.

Virological characterization of HIV-1 CA-NTD mutants constructed in a virus-lineage reflected manner.

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.

HIV-1 mutates to adapt in fluxing environments.

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.

Complete Genome Sequences of Human Immunodeficiency Type 1 Viruses Genetically Engineered To Be Tropic for Rhesus Macaques.

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 TRIM5α, APOBEC3, and tetherin proteins. The two HIV-1 variants described here are fundamental clones aiming for rhesus infection studies of HIV-1.

Generation and characterization of new CCR5-tropic HIV-1rmt clones.

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.