Phylogenetic Reconstruction and Functional Characterization of the Ancestral Nef Protein of Primate Lentiviruses.

Nef is an accessory protein unique to the primate HIV-1, HIV-2, and SIV lentiviruses. During infection, Nef functions by interacting with multiple host proteins within infected cells to evade the immune response and enhance virion infectivity. Notably, Nef can counter immune regulators such as CD4 and MHC-I, as well as the SERINC5 restriction factor in infected cells. In this study, we generated a posterior sample of time-scaled phylogenies relating SIV and HIV Nef sequences, followed by reconstruction of ancestral sequences at the root and internal nodes of the sampled trees up to the HIV-1 Group M ancestor. Upon expression of the ancestral primate lentivirus Nef protein within CD4+ HeLa cells, flow cytometry analysis revealed that the primate lentivirus Nef ancestor robustly downregulated cell-surface SERINC5, yet only partially downregulated CD4 from the cell surface. Further analysis revealed that the Nef-mediated CD4 downregulation ability evolved gradually, while Nef-mediated SERINC5 downregulation was recovered abruptly in the HIV-1/M ancestor. Overall, this study provides a framework to reconstruct ancestral viral proteins and enable the functional characterization of these proteins to delineate how functions could have changed throughout evolutionary history.

Different Patterns of Codon Usage and Amino Acid Composition across Primate Lentiviruses.

A common feature of the mammalian Lentiviruses (family Retroviridae) is an RNA genome that contains an extremely high frequency of adenine (31.7-38.2%) while being extremely poor in cytosine (13.9-21.2%). Such a biased nucleotide composition has implications for codon usage, causing a striking difference between the frequency of synonymous codons in Lentiviruses and that in their hosts. To test whether primate Lentiviruses present differences in codon and amino acid composition, we assembled a dataset of genome sequences that includes SIV species infecting Old-World monkeys and African apes, HIV-2, and the four groups of HIV-1. Using principal component analysis, we found that HIV-1 shows a significant enrichment in adenine plus thymine in the third synonymous codon position and in adenine and guanine in the first and second nonsynonymous codon positions. Similarly, we observed an enrichment in adenine and in guanine in nonsynonymous first and second codon positions, which affects the amino acid composition of the proteins Gag, Pol, Vif, Vpr, Tat, Rev, Env, and Nef. This result suggests an effect of natural selection in shaping codon usage. Under the hypothesis that the use of synonyms in HIV-1 could reflect adaptation to that of genes expressed in specific cell types, we found a highly significant correlation between codon usage in HIV-1 and monocytes, which was remarkably higher than that with B and T lymphocytes. This finding is in line with the notion that monocytes represent an HIV-1 reservoir in infected patients, and it could help understand how this reservoir is established and maintained.

Transcription Start Site Heterogeneity and Preferential Packaging of Specific Full-Length RNA Species Are Conserved Features of Primate Lentiviruses.

HIV-1 must package its RNA genome to generate infectious viruses. Recent studies have revealed that during genome packaging, HIV-1 not only excludes cellular mRNAs, but also distinguishes among full-length viral RNAs. Using NL4-3 and MAL molecular clones, multiple transcription start sites (TSS) were identified, which generate full-length RNAs that differ by only a few nucleotides at the 5' end. However, HIV-1 selectively packages RNAs containing one guanosine (1G RNA) over RNAs with three guanosines (3G RNA) at the 5' end. Thus, the 5' context of HIV-1 full-length RNA can affect its function. To determine whether the regulation of genome packaging by TSS usage is unique to NL4-3 and MAL, we examined 15 primate lentiviruses including transmitted founder viruses of HIV-1, HIV-2, and several simian immunodeficiency viruses (SIVs). We found that all 15 viruses used multiple TSS to some extent. However, the level of TSS heterogeneity in infected cells varied greatly, even among closely related viruses belonging to the same subtype. Most viruses also exhibited selective packaging of specific full-length viral RNA species into particles. These findings demonstrate that TSS heterogeneity and selective packaging of certain full-length viral RNA species are conserved features of primate lentiviruses. In addition, an SIV strain closely related to the progenitor virus that gave rise to HIV-1 group M, the pandemic pathogen, exhibited TSS usage similar to some HIV-1 strains and preferentially packaged 1G RNA. These findings indicate that multiple TSS usage and selective packaging of a particular unspliced RNA species predate the emergence of HIV-1. IMPORTANCE Unspliced HIV-1 RNA serves two important roles during viral replication: as the virion genome and as the template for translation of Gag/Gag-Pol. Previous studies of two HIV-1 molecular clones have concluded that the TSS usage affects unspliced HIV-1 RNA structures and functions. To investigate the evolutionary origin of this replication strategy, we determined TSS of HIV-1 RNA in infected cells and virions for 15 primate lentiviruses. All HIV-1 isolates examined, including several transmitted founder viruses, utilized multiple TSS and selected a particular RNA species for packaging. Furthermore, these features were observed in SIVs related to the progenitors of HIV-1, suggesting that these characteristics originated from the ancestral viruses. HIV-2, SIVs related to HIV-2, and other SIVs also exhibited multiple TSS and preferential packaging of specific unspliced RNA species, demonstrating that this replication strategy is broadly conserved across primate lentiviruses.

Analysis of evolutionary and genetic patterns in structural genes of primate lentiviruses.

BACKGROUND: Primate lentiviruses (HIV1, HIV2, and Simian immunodeficiency virus [SIV]) cause immune deficiency, encephalitis, and infectious anemia in mammals such as cattle, cat, goat, sheep, horse, and puma. OBJECTIVE: This study was designed and conducted with the main purpose of confirming the overall codon usage pattern of primate lentiviruses and exploring the evolutionary and genetic characteristics commonly or specifically expressed in HIV1, HIV2, and SIV. METHODS: The gag, pol, and env gene sequences of HIV1, HIV2, and SIV were analyzed to determine their evolutionary relationships, nucleotide compositions, codon usage patterns, neutrality, selection pressure (influence of mutational pressure and natural selection), and viral adaptation to human codon usage. RESULTS: A strong 'A' bias was confirmed in all three structural genes, consistent with previous findings regarding HIV. Notably, the ENC-GC3s plot and neutral evolution analysis showed that all primate lentiviruses were more affected by selection pressure than by mutation caused by the GC composition of the gene, consistent with prior reports regarding HIV1. The overall codon usage bias of pol was highest among the structural genes, while the codon usage bias of env was lowest. The virus groups showing high codon bias in all three genes were HIV1 and SIVcolobus. The codon adaptation index (CAI) and similarity D(A, B) values indicated that although there was a high degree of similarity to human codon usage in all three structural genes of HIV, this similarity was not caused by translation pressure. In addition, compared with HIV1, the codon usage of HIV2 is more similar to the human codon usage, but the overall codon usage bias is lower. CONCLUSION: The origin viruses of HIV (SIVcpz_gor and SIVsmm) exhibit greater similarity to human codon usage in the gag gene, confirming their robust adaptability to human codon usage. Therefore, HIV1 and HIV2 may have evolved to avoid human codon usage by selection pressure in the gag gene after interspecies transmission from SIV hosts to humans. By overcoming safety and stability issues, information from codon usage analysis will be useful for attenuated HIV1 vaccine development. A recoded HIV1 variant can be used as a vaccine vector or in immunotherapy to induce specific innate immune responses. Further research regarding HIV1 dinucleotide usage and codon pair usage will facilitate new approaches to the treatment of AIDS.

Primate lentiviruses require Inositol hexakisphosphate (IP6) or inositol pentakisphosphate (IP5) for the production of viral particles.

Inositol hexakisphosphate (IP6) potently stimulates HIV-1 particle assembly in vitro and infectious particle production in vivo. However, knockout cells lacking inositol-pentakisphosphate 2-kinase (IPPK-KO), the enzyme that produces IP6 by phosphorylation of inositol pentakisphosphate (IP5), were still able to produce infectious HIV-1 particles at a greatly reduced rate. HIV-1 in vitro assembly can also be stimulated to a lesser extent with IP5, but until recently, it was not known if IP5 could also function in promoting assembly in vivo. Here we addressed whether there is an absolute requirement for IP6 or IP5 in the production of infectious HIV-1 particles. IPPK-KO cells expressed no detectable IP6 but elevated IP5 levels and displayed a 20-100-fold reduction in infectious particle production, correlating with lost virus release. Transient transfection of an IPPK expression vector stimulated infectious particle production and release in IPPK-KO but not wildtype cells. Several attempts to make IP6/IP5 deficient stable cells were not successful, but transient expression of the enzyme multiple inositol polyphosphate phosphatase-1 (MINPP1) into IPPK-KOs resulted in near ablation of IP6 and IP5. Under these conditions, we found that HIV-1 infectious particle production and virus release were essentially abolished (1000-fold reduction) demonstrating an IP6/IP5 requirement. However, other retroviruses including a Gammaretrovirus, a Betaretrovirus, and two non-primate Lentiviruses displayed only a modest (3-fold) reduction in infectious particle production from IPPK-KOs and were not significantly altered by expression of IPPK or MINPP1. The only other retrovirus found to show a clear IP6/IP5 dependence was the primate (macaque) Lentivirus Simian Immunodeficiency Virus, which displayed similar sensitivity as HIV-1. We were not able to determine if producer cell IP6/IP5 is required at additional steps beyond assembly because viral particles devoid of both molecules could not be generated. Finally, we found that loss of IP6/IP5 in viral target cells had no effect on permissivity to HIV-1 infection.

Distinct MCM10 Proteasomal Degradation Profiles by Primate Lentiviruses Vpr Proteins.

Viral protein R (Vpr) is an accessory protein found in various primate lentiviruses, including human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) as well as simian immunodeficiency viruses (SIVs). Vpr modulates many processes during viral lifecycle via interaction with several of cellular targets. Previous studies showed that HIV-1 Vpr strengthened degradation of Mini-chromosome Maintenance Protein10 (MCM10) by manipulating DCAF1-Cul4-E3 ligase in proteasome-dependent pathway. However, whether Vpr from other primate lentiviruses are also associated with MCM10 degradation and the ensuing impact remain unknown. Based on phylogenetic analyses, a panel of primate lentiviruses Vpr/x covering main virus lineages was prepared. Distinct MCM10 degradation profiles were mapped and HIV-1, SIVmus and SIVrcm Vprs induced MCM10 degradation in proteasome-dependent pathway. Colocalization and interaction between MCM10 with these Vprs were also observed. Moreover, MCM10 2-7 interaction region was identified as a determinant region susceptible to degradation. However, MCM10 degradation did not alleviate DNA damage response induced by these Vpr proteins. MCM10 degradation by HIV-1 Vpr proteins was correlated with G2/M arrest, while induction of apoptosis and oligomerization formation of Vpr failed to alter MCM10 proteolysis. The current study demonstrated a distinct interplay pattern between primate lentiviruses Vpr proteins and MCM10.

Relationship between genome packaging and Gag translation/AUG of primate lentiviruses.

About the relationship between retroviral genome packaging and translation, three possible modes (random-, trans-, and cis-) of packaging process could be assumed. In this report, we developed an assay system based on the RT-qPCR to measure the packaging efficiency of primate lentiviruses. With this system, we analyzed the genome packaging modes of primate lentiviruses such as HIV-1, 2, SIVmac and SIVagm. The data suggested that the modes of all viruses analyzed were very similar. In addition, we observed that the Gag-AUG sequences of them played important roles for maintaining efficient packaging, other than the initiation of translation.

Primate immunodeficiency virus proteins Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex.

Host factors that silence provirus transcription in CD4+ memory T cells help HIV-1 escape eradication by the host immune system and by antiviral drugs1. These same factors, however, must be overcome for HIV-1 to propagate. Here we show that Vpx and Vpr encoded by diverse primate immunodeficiency viruses activate provirus transcription. Vpx and Vpr are adaptor proteins for the DCAF1-CUL4A/B E3 ubiquitin ligase that degrade SAMHD1 and increase reverse transcription2-4. Nonetheless, Vpx and Vpr have effects on reporter gene expression that are not explained by SAMHD1 degradation5-8. A screen for factors that mimic these effects identified the human silencing hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN) and MORC29-13. Vpx associated with the HUSH complex and decreased steady-state level of these proteins in a DCAF1/CUL4A/B/proteasome-dependent manner14,15. Replication kinetics of HIV-1 and SIVMAC was accelerated to a similar extent by vpx or FAM208A knockdown. Finally, vpx increased steady-state levels of LINE-1 ORF1p, as previously described for FAM208A disruption11. These results demonstrate that the HUSH complex represses primate immunodeficiency virus transcription, and that, to counteract this restriction, viral Vpx or Vpr proteins degrade the HUSH complex.

Resistance of Major Histocompatibility Complex Class B (MHC-B) to Nef-Mediated Downregulation Relative to that of MHC-A Is Conserved among Primate Lentiviruses and Influences Antiviral T Cell Responses in HIV-1-Infected Individuals.

Patient-derived HIV-1 subtype B Nef clones downregulate HLA-A more efficiently than HLA-B. However, it remains unknown whether this property is common to Nef proteins across primate lentiviruses and how antiviral immune responses may be affected. We examined 263 Nef clones from diverse primate lentiviruses including different pandemic HIV-1 group M subtypes for their ability to downregulate major histocompatibility complex class A (MHC-A) and MHC-B from the cell surface. Though lentiviral Nef proteins differed markedly in their absolute MHC-A and MHC-B downregulation abilities, all lentiviral Nef lineages downregulated MHC-A, on average, 11 to 32% more efficiently than MHC-B. Nef genotype/phenotype analyses in a cohort of HIV-1 subtype C-infected patients (n = 168), together with site-directed mutagenesis, revealed Nef position 9 as a subtype-specific determinant of differential HLA-A versus HLA-B downregulation activity. Nef clones harboring nonconsensus variants at codon 9 downregulated HLA-B (though not HLA-A) significantly better than those harboring the consensus sequence at this site, resulting in reduced recognition of infected target cells by HIV-1-specific CD8+ effector cells in vitro Among persons expressing protective HLA class I alleles, carriage of Nef codon 9 variants was also associated with reduced ex vivo HIV-specific T cell responses. Our results demonstrate that Nef's inferior ability to downregulate MHC-B compared to that of MHC-A is conserved across primate lentiviruses and suggest that this property influences antiviral cellular immune responses.IMPORTANCE Primate lentiviruses encode the Nef protein that plays an essential role in establishing persistent infection in their respective host species. Nef interacts with the cytoplasmic region of MHC-A and MHC-B molecules and downregulates them from the infected cell surface to escape recognition by host cellular immunity. Using a panel of Nef alleles isolated from diverse primate lentiviruses including pandemic HIV-1 group M subtypes, we demonstrate that Nef proteins across all lentiviral lineages downregulate MHC-A approximately 20% more effectively than MHC-B. We further identify a naturally polymorphic site at Nef position 9 that contributes to the MHC-B downregulation function in HIV-1 subtype C and show that carriage of Nef variants with enhanced MHC-B downregulation ability is associated with reduced breadth and magnitude of MHC-B-restricted cellular immune responses in HIV-infected individuals. Our study underscores an evolutionarily conserved interaction between lentiviruses and primate immune systems that may contribute to pathogenesis.

The HIV-1 Vpr Protein: A Multifaceted Target for Therapeutic Intervention.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein is an attractive target for antiretroviral drug development. The conservation both of the structure along virus evolution and the amino acid sequence in viral isolates from patients underlines the importance of Vpr for the establishment and progression of HIV-1 disease. While its contribution to virus replication in dividing and non-dividing cells and to the pathogenesis of HIV-1 in many different cell types, both extracellular and intracellular forms, have been extensively studied, its precise mechanism of action nevertheless remains enigmatic. The present review discusses how the apparently multifaceted interplay between Vpr and host cells may be due to the impairment of basic metabolic pathways. Vpr protein modifies host cell energy metabolism, oxidative status, and proteasome function, all of which are likely conditioned by the concentration and multimerization of the protein. The characterization of Vpr domains along with new laboratory tools for the assessment of their function has become increasingly relevant in recent years. With these advances, it is conceivable that drug discovery efforts involving Vpr-targeted antiretrovirals will experience substantial growth in the coming years.

Down-modulation of primate lentiviral receptors by Nef proteins of simian immunodeficiency virus (SIV) of chimpanzees (SIVcpz) and related SIVs: implication for the evolutionary event at the emergence of SIVcpz.

It has been estimated that human immunodeficiency virus type 1 originated from the zoonotic transmission of simian immunodeficiency virus (SIV) of chimpanzees, SIVcpz, and that SIVcpz emerged by the recombination of two lineages of SIVs in Old World monkeys (SIVgsn/mon/mus in guenons and SIVrcm in red-capped mangabeys) and SIVcpz Nef is most closely related to SIVrcm Nef. These observations suggest that SIVrcm Nef had an advantage over SIVgsn/mon/mus during the evolution of SIVcpz in chimpanzees, although this advantage remains uncertain. Nef is a multifunctional protein which downregulates CD4 and coreceptor proteins from the surface of infected cells, presumably to limit superinfection. To assess the possibility that SIVrcm Nef was selected by its superior ability to downregulate viral entry receptors in chimpanzees, we compared its ability to down-modulate viral receptor proteins from humans, chimpanzees and red-capped mangabeys with Nef proteins from eight other different strains of SIVs. Surprisingly, the ability of SIVrcm Nef to downregulate CCR5, CCR2B and CXCR6 was comparable to or lower than SIVgsn/mon/mus Nef, indicating that ability to down-modulate chemokine receptors was not the selective pressure. However, SIVrcm Nef significantly downregulates chimpanzee CD4 over SIVgsn/mon/mus Nefs. Our findings suggest the possibility that the selection of SIVrcm Nef by ancestral SIVcpz is due to its superior capacity to down-modulate chimpanzees CD4 rather than coreceptor proteins.

Involvement of a C-terminal motif in the interference of primate lentiviral Vpu proteins with CD1d-mediated antigen presentation.

The HIV-1 accessory protein Vpu is emerging as a critical factor for viral evasion from innate immunity. We have previously shown that the Vpu proteins of two HIV-1 group M subtype B strains (NL4-3 and BaL) down-regulate CD1d from the surface of infected dendritic cells (DCs) and inhibit their crosstalk with the innate invariant natural killer T (iNKT) cells. In the present study, we have investigated the ability of a comprehensive set of primate lentiviral Vpu proteins to interfere with CD1d-mediated immunity. We found that CD1d down-regulation is a conserved function of Vpu proteins from HIV-1 groups M, O and P as well as their direct precursors SIVcpzPtt and SIVgor. At the group M subtype level, subtype C Vpu proteins were significantly weaker CD1d antagonists than subtype B Vpu proteins. Functional characterization of different mutants and chimeras derived from active subtype B and inactive subtype C Vpu proteins revealed that residues in the cytoplasmic domain are important for CD1d down-regulation. Specifically, we identified a C-terminal APW motif characteristic for group M subtype B Vpu proteins necessary for interference with CD1d surface expression. These findings support the notion that Vpu plays an important role in lentiviral evasion from innate immunity.

Differential regulation of NF-κB-mediated proviral and antiviral host gene expression by primate lentiviral Nef and Vpu proteins.

NF-κB is essential for effective transcription of primate lentiviral genomes and also activates antiviral host genes. Here, we show that the early protein Nef of most primate lentiviruses enhances NF-κB activation. In contrast, the late protein Vpu of HIV-1 and its simian precursors inhibits activation of NF-κB, even in the presence of Nef. Although this effect of Vpu did not correlate with its ability to interact with ß-TrCP, it involved the stabilization of IκB and reduced nuclear translocation of p65. Interestingly, however, Vpu did not affect casein kinase II-mediated phosphorylation of p65. Lack of Vpu was associated with increased NF-κB activation and induction of interferon and interferon-stimulated genes (ISGs) in HIV-1-infected T cells. Thus, HIV-1 and its simian precursors employ Nef to boost NF-κB activation early during the viral life cycle to initiate proviral transcription, while Vpu is used to downmodulate NF-κB-dependent expression of ISGs at later stages.

A primitive endogenous lentivirus in a colugo: insights into the early evolution of lentiviruses.

Lentiviruses infect a wide range of mammal species. Much remains unknown about their deep history and host distribution. Here, we report the discovery of an endogenous lentivirus within the genome of the Sunda flying lemur (Galeopterus variegatus) (which we designate "Galeopterus variegatus endogenous lentivirus" [GvaELV]). We estimate the GvaELV genome invasion to have occurred more than 14 Ma, supporting an ancient origin of the lentivirus clade and an ancient lentiviral infection in colugo. Phylogenetic analyses show that GvaELV is a sister group of all previously known lentiviruses. The GvaELV genome appears to possess some primitive genomic features of a lentivirus, encoding not only a trans-activator of transcription (tat) gene but also two additional putative accessory genes that share no discernible similarity with other lentiviral accessory genes. The discovery of GvaELV provides novel insights into the prehistory and host distribution of lentivirus.

The ability of primate lentiviruses to degrade the monocyte restriction factor SAMHD1 preceded the birth of the viral accessory protein Vpx.

The human SAMHD1 protein potently restricts lentiviral infection in dendritic cells and monocyte/macrophages but is antagonized by the primate lentiviral protein Vpx, which targets SAMHD1 for degradation. However, only two of eight primate lentivirus lineages encode Vpx, whereas its paralog, Vpr, is conserved across all extant primate lentiviruses. We find that not only multiple Vpx but also some Vpr proteins are able to degrade SAMHD1, and such antagonism led to dramatic positive selection of SAMHD1 in the primate subfamily Cercopithecinae. Residues that have evolved under positive selection precisely determine sensitivity to Vpx/Vpr degradation and alter binding specificity. By overlaying these functional analyses on a phylogenetic framework of Vpr and Vpx evolution, we can decipher the chronology of acquisition of SAMHD1-degrading abilities in lentiviruses. We conclude that vpr neofunctionalized to degrade SAMHD1 even prior to the birth of a separate vpx gene, thereby initiating an evolutionary arms race with SAMHD1.

Down-modulation of CD8αß is a fundamental activity of primate lentiviral Nef proteins.

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αß (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8ß in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8ß, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8ß, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αß is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.

Antibodies and lentiviruses that specifically recognize a T cell epitope derived from HIV-1 Nef protein and presented by HLA-C.

HIV selectively downregulates HLA-A and -B from the surfaces of infected cells to avoid detection by the immune system. In contrast, the HLA-C molecules are highly resistant to this downregulation. High expression level of HLA-C on the cell surface, which correlates with a single nucleotide polymorphism, is also associated with lower viral loads and slower progression to AIDS. These findings strongly suggest that HIV-1-derived peptides are efficiently presented by HLA-C and trigger the elimination of infected cells. Accordingly, the ability to detect these HLA-C-peptide complexes may be used for therapeutic targeting of HIV-1-infected cells and for measuring effective presentation of vaccine candidates after immunization with HIV-1-related proteins or genes. However, low level of HLA-C expression on the cell surface has impeded the development of such complex-recognizing reagents. In this study, we describe the development of a high-affinity human Ab that specifically interacts, at low pM concentrations, with a conserved viral T cell epitope derived from HIV-1 Nef protein and presented by HLA-C. The human Ab selectively detects this complex on different cells and does not interact with a control complex that differed only in the presented peptide. Engineering lentiviruses to display this Ab endowed them with the same specificity as the Ab, whereas coexpressing the Ab and Fas ligand enables the lentiviruses to kill specifically Nef-presenting cells. Abs and pseudoviruses with such specificity are likely to be highly valuable as building blocks for specific targeting and killing of HIV-1-infected cells.

Structural and functional analysis of prehistoric lentiviruses uncovers an ancient molecular interface.

Lentiviruses are widespread in a variety of vertebrates, often associated with chronic disease states. However, until the recent discovery of the prehistoric endogenous lentiviruses in rabbits (RELIK) and lemurs (PSIV), it was thought that lentiviruses had no capacity for germline integration and were only spread horizontally in an exogenous fashion. The existence of RELIK and PSIV refuted these ideas, revealing lentiviruses to be present in a range of mammals, capable of germline integration, and far more ancient than previously thought. Using Gag sequences reconstructed from the remnants of these prehistoric lentiviruses, we have produced chimeric lentiviruses capable of infecting nondividing cells and determined structures of capsid domains from PSIV and RELIK. We show that the structures from these diverse viruses are highly similar, containing features found in modern-day lentiviruses, including a functional cyclophilin-binding loop. Together, these data provide evidence for an ancient capsid-cyclophilin interaction preserved throughout lentiviral evolution.

BST-2/tetherin: a new component of the innate immune response to enveloped viruses.

The interferon-inducible, transmembrane protein BST-2 (CD317, tetherin) directly holds fully formed enveloped virus particles to the cells that produce them, inhibiting their spread. BST-2 inhibits members of the retrovirus, filovirus, arenavirus and herpesvirus families. These viruses encode a variety of proteins to degrade BST-2 and/or direct it away from its site of action at the cell surface. Viral antagonism has subjected BST-2 to positive selection, leading to species-specific differences that presented a barrier to the transmission of simian immunodeficiency viruses (SIVs) to humans. This barrier was crossed by HIV-1 when its Vpu protein acquired activity as a BST-2 antagonist. Here, we review this new host-pathogen relationship and discuss its impact on the evolution of primate lentiviruses and the origins of the HIV pandemic.

Ancient adaptive evolution of tetherin shaped the functions of Vpu and Nef in human immunodeficiency virus and primate lentiviruses.

Tetherin/BST-2 is a host-encoded protein that restricts a wide diversity of viruses at the stage of virion release. However, viruses have evolved antagonists of Tetherin, including the Vpu and Nef proteins of primate lentiviruses. Like other host genes subject to viral antagonism, primate Tetherin genes have evolved under positive selection. We show here that viral antagonists acting at three independent sites of selection have driven the evolution of Tetherin, with the strongest selective pressure on the cytoplasmic tail domain. Human Tetherin is unique among the Tetherins of simian primates in that it has a 5-amino-acid deletion that results in the loss of the residue under the strongest positive selection. We show that this residue at amino acid 17 is the site of the functional interaction of Tetherin with Nef, since single amino acid substitutions at this single position can determine the susceptibility of Tetherin to Nef antagonism. While the simian immunodeficiency viruses SIVcpz and SIVgor are able to antagonize their hosts' Tetherin with Nef, human immunodeficiency virus type 1 (HIV-1) Vpu has evolved to counteract Tetherin in humans. We mapped the adaptations in the N-terminal transmembrane domain of Vpu that allow it to counteract human Tetherin. Our combined evolutionary and functional studies have allowed us to reconstruct the host-pathogen interactions that have shaped Tetherin and its lentivirus-encoded antagonists.