Naoya Doi

Species barrier of HIV-1 and its jumping by virus engineering

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 5α (TRIM5α) 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. Copyright

Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/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 TRIM5α 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 TRIM5α. 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.

Systemic biological analysis of the mutations in two distinct HIV-1mt genomes occurred during replication in macaque cells

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.

In silico Analysis of HIV-1 Env-gp120 Reveals Structural Bases for Viral Adaptation in Growth-Restrictive Cells

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.

Natural Single-Nucleotide Polymorphisms in the 3′ Region of the HIV-1 pol Gene Modulate Viral Replication Ability

ABSTRACT We previously showed that prototype macaque-tropic human immunodeficiency virus type 1 (HIV-1) acquired nonsynonymous growth-enhancing mutations within a narrow genomic region during the adaptation process in macaque cells. These adaptive mutations were clustered in the 3′ region of the pol gene, encoding a small portion of the C-terminal domain of integrase (IN). Mutations in HIV-1 IN have been reported to have pleiotropic effects on both the early and late phases in viral replication. cis-acting functions in the IN-coding sequence for viral gene expression have also been reported. We here demonstrated that the adaptive mutations promoted viral growth by increasing virion production with no positive effects on the early replication phase. Synonymous codon alterations in one of the adaptive mutations influenced virion production levels, which suggested nucleotide-dependent regulation. Indeed, when the single-nucleotide natural polymorphisms observed in the 3′ regions of 196 HIV-1/simian immunodeficiency virus (SIVcpz) pol genes (nucleotides [nt] 4895 to 4929 for HIV-1 NL4-3) were introduced into macaque- and human-tropic HIV-1 clones, more than half exhibited altered replication potentials. Moreover, single-nucleotide mutations caused parallel increases or decreases in the expression levels of viral late proteins and viral replication potentials. We also showed that the overall expression profiles of viral mRNAs were markedly changed by single-nucleotide mutations. These results demonstrate that the 3′ region of the HIV-1 pol gene (nt 4895 to 4929) can alter viral replication potential by modulating the expression pattern of viral mRNAs in a nucleotide-dependent manner. IMPORTANCE 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.

Poly-proline motif in HIV-2 Vpx is critical for its efficient translation

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.

Rhesus M1.3S Cells Suitable for Biological Evaluation of Macaque-Tropic HIV/SIV Clones

Human immunodeficiency virus type 1 (HIV-1) is the causative virus of human acquired immunodeficiency syndrome (AIDS). Due to the lack of appropriate animal models, basic studies on HIV-1 replication, pathogenesis, and evolution, have been limited to cellular and molecular levels (Nomaguchi et al., 2008). Moreover, applied clinical studies in vivo also have been forced to use simian immunodeficiency virus isolated from macaques (SIVmac) and/or chimeric virus between SIVmac and HIV-1 (SHIV) based on our pioneering work 20 years ago (Shibata et al., 1991; Sakuragi et al., 1992). This experimental hindrance is mainly originated from the extremely narrow host range of HIV-1. To overcome the difficulty, we and others have made every effort to develop and establish suitable virus/animal systems. Because HIV-1 has adapted itself from the ancestral SIVs in Africa to replicate in humans in a marvelously strict way (Kirchhoff, 2009), the most promising one would be the use of macaque-tropic HIV-1 and macaques (Ambrose et al., 2007; Nomaguchi et al., 2008). As input viruses for infection of macaques, simian-tropic (st)HIV-1, and macaque-tropic HIV-1 (HIV-1mt) were designed, generated and characterized in vitro (Hatziioannou et al., 2006; Kamada et al., 2006; Thippeshappa et al., 2011) and in vivo (Igarashi et al., 2007; Hatziioannou et al., 2009; Saito et al., 2011; Thippeshappa et al., 2011). However, any of these HIV-1 derivatives neither replicate similarly robustly with the SIVmac standard clone in macaque cells nor are pathogenic for macaques so far. We, therefore, started improving the prototype HIV-1mt clone designated NL-DT5R by viral adaptation in macaque cells and genome manipulation based on bioinformatics (Figure ​(Figure1A).1A). NL-DT5R contains a 21-nucleotide SIVmac Gag-capsid (CA) element that lacks cyclophilin A (CypA) binding activity of the corresponding HIV-1 Gag-CA loop [CypA binding loop (−) in Figure ​Figure1A]1A] and the entire SIVmac vif gene (Kamada et al., 2006). NL-DT5RS contains a loop sequence between helices 6 and 7 (h6/7L in Figure ​Figure1A)1A) of SIVmac Gag-CA relative to NL-DT5R. MN4–8 carries three adaptive mutations in the pol-integrase and env-gp120 regions. MN4–8S contains the h6/7L of SIVmac Gag-CA and three adaptive mutations. MN4Rh-3 has an additional mutation in Gag-CA relative to MN4–8S. We now have a newest clone MN4Rh-3V (CXCR4-tropic), which was generated by the introduction of several mutations deduced from structural analysis into Gag-CA. Because CCR5-tropic viruses are thought to be clinically more important than CXCR4-tropic viruses, we similarly have generated a CCR5-tropic clone designated MN5Rh-3V. Figure 1 Virological characteristics of HIV-1/SIVmac clones. (A) Proviral genome structure of various viruses used for infection of M1.3S. Genomes of various HIV-1mt clones generated in our laboratory are schematically illustrated. Parental clones HIV-1 NL4-3 ... To achieve our purpose to obtain HIV-1mt clones potentially pathogenic for macaques, as a prerequisite, it is critical to have macaque cell lines suitable and easily usable to characterize many of the virus clones generated. However, macaque cell lines that are sensitive to various SIV and HIV-1mt clones and maintain the characteristics of natural target cells such as peripheral blood mononuclear cells were not readily available to us. We therefore searched for the appropriate cell lines and finally paid attention to the macaque cell lines immortalized by Herpesvirus saimiri (Akari et al., 1996; Fujita et al., 2003; Doi et al., 2010). Of various cynomolgus and rhesus macaque cell lines in these reports, we were particularly interested in the rhesus MT-IL2I cell line (Doi et al., 2010). It is the most refractory cell line among those examined by us to infection of SIVmac and HIV-1mt clones, but is CD4-positive, CXCR4-positive, and CCR5-positive. Therefore, it was predicted that this cell line expresses potent intracellular restriction factors against viruses. In fact, the TRIM5 allele of this cell line was found to be Mamu 1/Mamu 3 (Doi et al., 2010), a genotype that phenotypically shows a strong resistance to SIVmac infection (Lim et al., 2010). A subline of the MT-IL2I, after CD4-positive cells had been enriched by sorting, was designated M1.3S and used for infection experiments thereafter. Representative results obtained in M1.3S are shown in Figure ​Figure1B.1B. Viruses of the early generation (from the prototype NL-DT5R up to MN4–8S) did not replicate at all. However, in cynomolgus HSC-F cells that are very sensitive to virus infection, MN4–8S replicated better than NL-DT5R, NL-DT5RS, and MN4–8. Interestingly, MN4Rh-3 acquired replication ability in M1.3S cells, and MN4Rh-3V exhibited further improved replication potential (Figure ​(Figure1B1B left). On the other hand, the mutational effects of SIVmac accessory proteins on viral replication are also readily observed in M1.3S cells (Figure ​(Figure1B1B right). Generally, these effects cannot be easily recognized in the established cell lines. As clearly observed in Figure ​Figure1B,1B, each mutation to knock out the expression of individual accessory protein has greatly affected SIVmac replication. In particular, the mutations in vif and vpx genes have demonstrated a drastic negative effect. Therefore, it is quite evident that restriction factors against virus replication (counteracted by viral accessory proteins) are amply present in M1.3S cells. In conclusion, the M1.3S cell line is exquisitely suitable to characterize macaque-tropic viruses and to investigate anti-viral cellular factors. Although primary cells are indispensable for basic studies on HIV/SIV that have highly adapted themselves to replicate in these cells, M1.3S cells can alternatively be used to address important issues to understand the biology and molecular biology of HIV/SIV.

Macaque-Tropic HIV-1 Derivatives: A Novel Experimental Approach to Understand Viral Replication and Evolution in Vivo

The use of animal models in the study of human diseases is obviously important. Fundamental properties of the disease can be investigated analytically and thoroughly by this approach, contributing much to the progress of basic science as well as clinical medicine (Nomaguchi & Adachi, 2010). Researchers in various specialties, therefore, have made every effort to establish animal models for human diseases including those caused by infectious agents. Acquired immunodeficiency syndrome (AIDS) of humans has long been one of the major targets for the model study in appropriate animals. However, human immunodeficiency virus type 1 (HIV-1) itself, the predominant causative virus of human AIDS, can not be used due to its very narrow host range. Because HIV-1 has adapted itself dexterously from the ancestral virus to replicate, persist and spread strictly in humans, it is very unique among various primate immunodeficiency viruses and no good counterparts are available in nature (Desrosiers, 2007; Kirchhoff, 2009; Sauter et al., 2009). Therefore, it can be concluded that practical and meaningful animal systems of non-alternative nature for HIV-1 study do not exist at all to date, although there are pre-existing animal models of some significance. HIV-1 does not replicate in animal species except for chimpanzees and humans (Nomaguchi et al., 2008a). Animals frequently used for our experiments on virology, such as rodents and nonhuman primates, are not exceptions to this barrier. However, if we are to search for, develop and establish a fruitful animal model system for HIV-1 research, nonhuman primates are considered to be most suited, for HIV-1 is best fitted with humans and some apes. Ever since the discovery of HIV-1 (Barre-Sinoussi et al., 1983), many prominent researchers keen on understanding its biology and molecular biology have done investigations extensively to elucidate the bases underlying the species-specificity unique to HIV-1. These studies have highlighted the presence of potent anti-HIV-1 factors in nonhuman cells that efficiently restrict or even abolish the replication of HIV-1 and successfully raised an epoch-making notion of the intrinsic immunity (Andrew & Strebel, 2010; Arhel & Kirchhoff, 2010; Ayinde et al., 2010; Bergamaschi & Pancino, 2010; Douglas et al., 2010; Fujita et al., 2010; Huthoff & Towers, 2008; Kirchhoff, 2010; Luban, 2007; Malim & Emerman, 2008; Nakayama & Shioda, 2010; Nomaguchi et al., 2008a, 2008b; Planelles & Barker, 2010; Sauter et al., 2010; Strebel et al., 2009; Towers, 2007). Cellular factors shoulder

Analysis of the cell-dependent replication potentials of human immunodeficiency virus type 1 vif mutants

Eleven in-frame vif gene mutants of HIV type 1 produced in non-permissive cells were examined for their replication potentials in various CD4-positive and -negative cell lines. Virus replication for each mutant was monitored by using several single- and multiple-cycle infectivity assays. Except for a mutant with wild-type phenotype, most mutants were severely defective for replication in all the cell lines as expected from the producer cell-dependent functioning of Vif so far reported. In contrast, two mutants, which have mutations in the hydrophilic or effector regions of Vif were found to have target cell-dependent replication potentials. These results demonstrate the presence of a novel category of the vif mutants important for elucidation of the Vif function.

Distinct combinations of amino acid substitutions in N-terminal domain of Gag-capsid afford HIV-1 resistance to rhesus TRIM5α.

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.