Recombinant origin and interspecies transmission of a HERV-K(HML-2)-related primate retrovirus with a novel RNA transport element.

HERV-K(HML-2), the youngest clade of human endogenous retroviruses (HERVs), includes many intact or nearly intact proviruses, but no replication competent HML-2 proviruses have been identified in humans. HML-2-related proviruses are present in other primates, including rhesus macaques, but the extent and timing of HML-2 activity in macaques remains unclear. We have identified 145 HML-2-like proviruses in rhesus macaques, including a clade of young, rhesus-specific insertions. Age estimates, intact open reading frames, and insertional polymorphism of these insertions are consistent with recent or ongoing infectious activity in macaques. 106 of the proviruses form a clade characterized by an ~750 bp sequence between env and the 3' long terminal repeat (LTR), derived from an ancient recombination with a HERV-K(HML-8)-related virus. This clade is found in Old World monkeys (OWM), but not great apes, suggesting it originated after the ape/OWM split. We identified similar proviruses in white-cheeked gibbons; the gibbon insertions cluster within the OWM recombinant clade, suggesting interspecies transmission from OWM to gibbons. The LTRs of the youngest proviruses have deletions in U3, which disrupt the Rec Response Element (RcRE), required for nuclear export of unspliced viral RNA. We show that the HML-8-derived region functions as a Rec-independent constitutive transport element (CTE), indicating the ancestral Rec-RcRE export system was replaced by a CTE mechanism.

Just as we study fossils to understand how animals and plants have evolved, we can study ancient viruses to understand how diseases have emerged and changed over long periods. Unlike fossils, viruses do not leave visible traces in the ground but, instead, they leave viral genes known as endogenous viral elements (or EVEs) that become permanently incorporated in their host's DNA. HML-2s are the youngest known EVEs in the human genome. They have evolved gradually by accumulating lots of small genetic changes and no longer actively infect humans. But these virus remnants have long been suspected to play a role in prostate cancer, lupus and other human diseases. Rhesus macaques and other monkeys also have HML-2s but these are less well studied than human HML-2s. Monkeys are often used as models of human biology in research studies, therefore, understanding how HML-2s have evolved in rhesus macaques may enable researchers to establish this monkey as a model for investigating the role of HML-2s in humans. To investigate this possibility, Williams et al. searched for HML-like EVEs in rhesus macaque genomes published in previous studies. The experiments found that, unlike human HML-2s, the macaque HML-2s underwent a sudden genetic transformation millions of years ago. They acquired a new gene from another virus that completely changed how the macaque HML-2s leave a compartment within the cells of their host that contains most of the host's genome ­ a key step in the life cycle of viruses. The data also suggest that HML-2s may still be actively infecting macaques today and that these EVEs jumped from monkeys into gibbons. This is the first known example of HML-2s moving between different types of primates and it indicates there may be a risk that macaque HML-2s could infect humans. In the future, the findings of Williams et al. may help researchers develop new approaches to treat prostate cancer and other diseases linked with HML-2s in humans.

Unique Structure and Distinctive Properties of the Ancient and Ubiquitous Gamma-Type Envelope Glycoprotein.

After the onset of the AIDS pandemic, HIV-1 (genus Lentivirus) became the predominant model for studying retrovirus Env glycoproteins and their role in entry. However, HIV Env is an inadequate model for understanding entry of viruses in the Alpharetrovirus, Gammaretrovirus and Deltaretrovirus genera. For example, oncogenic model system viruses such as Rous sarcoma virus (RSV, Alpharetrovirus), murine leukemia virus (MLV, Gammaretrovirus) and human T-cell leukemia viruses (HTLV-I and HTLV-II, Deltaretrovirus) encode Envs that are structurally and functionally distinct from HIV Env. We refer to these as Gamma-type Envs. Gamma-type Envs are probably the most widespread retroviral Envs in nature. They are found in exogenous and endogenous retroviruses representing a broad spectrum of vertebrate hosts including amphibians, birds, reptiles, mammals and fish. In endogenous form, gamma-type Envs have been evolutionarily coopted numerous times, most notably as placental syncytins (e.g., human SYNC1 and SYNC2). Remarkably, gamma-type Envs are also found outside of the Retroviridae. Gp2 proteins of filoviruses (e.g., Ebolavirus) and snake arenaviruses in the genus Reptarenavirus are gamma-type Env homologs, products of ancient recombination events involving viruses of different Baltimore classes. Distinctive hallmarks of gamma-type Envs include a labile disulfide bond linking the surface and transmembrane subunits, a multi-stage attachment and fusion mechanism, a highly conserved (but poorly understood) "immunosuppressive domain", and activation by the viral protease during virion maturation. Here, we synthesize work from diverse retrovirus model systems to illustrate these distinctive properties and to highlight avenues for further exploration of gamma-type Env structure and function.

Structure of the Receptor Binding Domain of EnvP(b)1, an Endogenous Retroviral Envelope Protein Expressed in Human Tissues.

EnvP(b)1 is an endogenous retroviral envelope gene found in human and other primate genomes. We report EnvP(b)1 sequences in primate genomes consistent with an integration event between 40 and 71 million years ago. Using a highly specific polyclonal antiserum raised against the putative receptor binding domain (RBD) of human EnvP(b)1, we detected expression in human placenta, ovaries, and thymus. We found that EnvP(b)1 is proteolytically processed, and using cell-cell fusion assays in multiple primate cell lines, we demonstrated that extant EnvP(b)1 proteins from a variety of primate genomes are fusogenic. This work supports the idea that EnvP(b)1 is under purifying selection and its fusogenic activity has been maintained for over 40 million years. We determined the structure of the RBD of human EnvP(b)1, which defines structural similarities with extant leukemia viruses, despite little sequence conservation. This structure highlights a common scaffold from which novel receptor binding specificities likely evolved. The evolutionary plasticity of this domain may underlie the diversity of related Envs in circulating viruses.IMPORTANCE Organisms can access genetic and functional novelty by capturing viral elements within their genomes, where they can evolve to drive new cellular or organismal processes. We demonstrate that a retroviral envelope gene, EnvP(b)1, has been maintained and its fusion activity preserved for 40 to 71 million years. It is expressed as a protein in multiple healthy human tissues. We determined the structure of its inferred receptor binding domain and compared it with the same domain in modern viruses. We found a common conserved architecture that underlies the varied receptor binding activity of divergent Env genes. The modularity and versatility of this domain may underpin the evolutionary success of this clade of fusogens.

Complex Problems Require Ternary Solutions: Another Lesson from SIV Nef.

In this issue of Cell Host & Microbe, Buffalo et al. describe a cryo-EM structure of SIV Nef complexed with AP-2 and tetherin. The structure helps explain why human tetherin is Nef-resistant and why lentiviruses that successfully emerged in humans (HIV-1 and HIV-2) had to evolve novel anti-tetherin strategies.

Origins and evolutionary consequences of ancient endogenous retroviruses.

Retroviruses infect a broad range of vertebrate hosts that includes amphibians, reptiles, fish, birds and mammals. In addition, a typical vertebrate genome contains thousands of loci composed of ancient retroviral sequences known as endogenous retroviruses (ERVs). ERVs are molecular remnants of ancient retroviruses and proof that the ongoing relationship between retroviruses and their vertebrate hosts began hundreds of millions of years ago. The long-term impact of retroviruses on vertebrate evolution is twofold: first, as with other viruses, retroviruses act as agents of selection, driving the evolution of host genes that block viral infection or that mitigate pathogenesis, and second, through the phenomenon of endogenization, retroviruses contribute an abundance of genetic novelty to host genomes, including unique protein-coding genes and cis-acting regulatory elements. This Review describes ERV origins, their diversity and their relationships to retroviruses and discusses the potential for ERVs to reveal virus-host interactions on evolutionary timescales. It also describes some of the many examples of cellular functions, including protein-coding genes and regulatory elements, that have evolved from ERVs.

High-Resolution Sequencing of Viral Populations during Early Simian Immunodeficiency Virus Infection Reveals Evolutionary Strategies for Rapid Escape from Emerging Env-Specific Antibody Responses.

Primate lentiviruses, including the human and simian immunodeficiency viruses (HIV and SIV), produce infections marked by persistent, ongoing viral replication. This occurs despite the presence of virus-specific adaptive immune responses, including antibodies targeting the viral envelope glycoprotein (Env), and evolution of antibody-escape variants is a well-documented feature of lentiviral infection. Here, we examined the evolutionary dynamics of the SIV env gene during early infection (≤29 weeks postinfection) in a cohort of four SIVmac251-infected rhesus macaques. We tracked env evolution during acute and early infection using frequent sampling and ultradeep sequencing of viral populations, capturing a transmission bottleneck and the subsequent reestablishment of Env diversity. A majority of changes in the gp120 subunit mapped to two short clusters, one in the first variable region (V1) and one in V4, while most changes in the gp41 subunit appeared in the cytoplasmic domain. Variation in V1 was dominated by short duplications and deletions of repetitive sequence, while variation in V4 was marked by short in-frame deletions and closely overlapping substitutions. The most common substitutions in both patches did not alter viral replicative fitness when tested using a highly sensitive, deep-sequencing-based competition assay. Our results, together with the observation that very similar or identical patterns of sequence evolution also occur in different macaque species infected with related but divergent strains of SIV, suggest that resistance to early, strain-specific anti-Env antibodies is the result of temporally and mutationally predictable pathways of escape that occur during the early stages of infection.IMPORTANCE The envelope glycoprotein (Env) of primate lentiviruses mediates entry by binding to host cell receptors followed by fusion of the viral membrane with the cell membrane. The exposure of Env complexes on the surface of the virion results in targeting by antibodies, leading to selection for virus escape mutations. We used the SIV/rhesus macaque model to track in vivo evolution of variation in Env during acute/early infection in animals with and without antibody responses to Env, uncovering remarkable variation in animals with antibody responses within weeks of infection. Using a deep-sequencing-based fitness assay, we found substitutions associated with antibody escape had little to no effect on inherent replicative capacity. The ability to readily propagate advantageous changes that incur little to no replicative fitness costs may be a mechanism to maintain continuous replication under constant immune selection, allowing the virus to persist for months to years in the infected host.

Retroviruses of the RDR superinfection interference group: ancient origins and broad host distribution of a promiscuous Env gene.

Due to recombination, different regions of a retrovirus genome can have distinct phylogenetic histories. The RD114-and-D-type-retrovirus (RDR) interference group provides an extreme example: the RDR group comprises a variety of taxonomically distinct retroviruses, isolated from diverse mammalian and avian hosts, that share a homologous env gene and use the same cell-surface entry receptor. RDR env homologs are also found among ancient endogenous retrovirus (ERV) sequences, including the syncytin genes of humans and rabbits, indicating that RDR Env glycoproteins have likely mediated endogenization on multiple occasions in diverse vertebrate lineages. The distribution of RDR env among exogenous and endogenous retroviruses indicates that it has been swapped between viruses many times, and that it likely facilitated multiple cross-species transmission events spanning millions of years of vertebrate evolution.

A penalized regression approach to haplotype reconstruction of viral populations arising in early HIV/SIV infection.

MOTIVATION: Next generation sequencing (NGS) has been increasingly applied to characterize viral evolution during HIV and SIV infections. In particular, NGS datasets sampled during the initial months of infection are characterized by relatively low levels of diversity as well as convergent evolution at multiple loci dispersed across the viral genome. Consequently, fully characterizing viral evolution from NGS datasets requires haplotype reconstruction across large regions of the viral genome. Existing haplotype reconstruction algorithms have not been developed with the particular characteristics of early HIV/SIV infection in mind, raising the possibility that better performance could be achieved through a specifically designed algorithm. RESULTS: Here, we introduce a haplotype reconstruction algorithm, RegressHaplo, specifically designed for low diversity and convergent evolution regimes. The algorithm uses a penalized regression that balances a data fitting term with a penalty term that encourages solutions with few haplotypes. The regression covariates are a large set of potential haplotypes and fitting the regression is made computationally feasible by the low diversity setting. Using simulated and in vivo datasets, we compare RegressHaplo to PredictHaplo and QuRe, two existing haplotype reconstruction algorithms. RegressHaplo performs better than these algorithms on simulated datasets with relatively low diversity levels. We suggest RegressHaplo as a novel tool for the investigation of early infection HIV/SIV datasets and, more generally, low diversity viral NGS datasets. CONTACT: sr286@georgetown.edu. AVAILABILITY AND IMPLEMENTATION: https://github.com/SLeviyang/RegressHaplo.

An Intact Retroviral Gene Conserved in Spiny-Rayed Fishes for over 100 My.

We have identified a retroviral envelope gene with a complete, intact open reading frame (ORF) in 20 species of spiny-rayed fishes (Acanthomorpha). The taxonomic distribution of the gene, "percomORF", indicates insertion into the ancestral lineage >110 Ma, making it the oldest known conserved gene of viral origin in a vertebrate genome. Underscoring its ancient provenence, percomORF exists as an isolated ORF within the intron of a widely conserved host gene, with no discernible proviral sequence nearby. Despite its remarkable age, percomORF retains canonical features of a retroviral glycoprotein, and tests for selection strongly suggest cooption for a host function. Retroviral envelope genes have been coopted for a role in placentogenesis by numerous lineages of mammals, including eutherians and marsupials, representing a variety of placental structures. Therefore percomORF's presence within the group Percomorpha-unique among spiny-finned fishes in having evolved placentation and live birth-is especially intriguing.

Analysis of SIVmac Envelope-Specific Antibodies Selected Through Phage Display.

We have constructed a single chain fragment variable (scFv) phage display library from a simian immunodeficiency virus (SIV)-infected rhesus macaque that developed unusually high-titer neutralizing antibody responses against tier-3, neutralization-resistant SIVmac239. The library was screened using trimeric (gp140) and monomeric (gp120) forms of the SIVmac239 envelope (Env) glycoprotein. We also cloned variable-heavy and variable-light (VH-VL) antibody fragments from seven previously described rhesus macaque B-cell lines (BLCLs) that produce SIV gp120-specific monoclonal antibodies (mAbs). Thirty-two gp140-specific mAbs were selected along with 20 gp120-specific ones. gp120-specific mAbs were only from the VH4 family, while gp41-specific mAbs were primarily from VH1, followed by VH4 and VH3. Rhesus macaque BLCL-derived mAbs belonged primarily to the VH4 family of antibodies followed by VH3 and a smaller number of VH1s. A preferential VH combination with Vλ light chain was observed with phage display-selected SIV Env-specific mAbs (gp120 and gp140), but not with BLCL-derived antibodies or the unpanned library. None of the tested antibodies had detectable neutralizing activity against tier-3 SIVmac239. The majority of gp120-specifc mAbs potently neutralized tier-1 SIVmac316 with 50% inhibitory concentration (IC50) values below 1 µg/ml. For gp140-specific antibodies, which were all specific for the gp41-subunit, 2 out of 11 tested neutralized SIVmac316 (IC50 of 7 and 5 µg/ml, respectively). These data suggest an order of preferential VH segment usage for SIV-specific antibodies in rhesus macaques. These antibodies will be useful in assessing the contribution of non-neutralizing antibodies to inhibition of SIV infection in vitro and in vivo.

Phylogeny and History of the Lost SIV from Crab-Eating Macaques: SIVmfa.

In the 20th century, thirteen distinct human immunodeficiency viruses emerged following independent cross-species transmission events involving simian immunodeficiency viruses (SIV) from African primates. In the late 1900s, pathogenic SIV strains also emerged in the United Sates among captive Asian macaque species following their unintentional infection with SIV from African sooty mangabeys (SIVsmm). Since their discovery in the 1980s, SIVs from rhesus macaques (SIVmac) and pig-tailed macaques (SIVmne) have become invaluable models for studying HIV pathogenesis, vaccine design and the emergence of viruses. SIV isolates from captive crab-eating macaques (SIVmfa) were initially described but lost prior to any detailed molecular and genetic characterization. In order to infer the origins of the lost SIVmfa lineage, we located archived material and colony records, recovered its genomic sequence by PCR, and assessed its phylogenetic relationship to other SIV strains. We conclude that SIVmfa is the product of two cross-species transmission events. The first was the established transmission of SIVsmm to rhesus macaques, which occurred at the California National Primate Research Center in the late 1960s and the virus later emerged as SIVmac. In a second event, SIVmac was transmitted to crab-eating macaques, likely at the Laboratory for Experimental Medicine and Surgery in Primates in the early 1970s, and it was later spread to the New England Primate Research Center colony in 1973 and eventually isolated in 1986. Our analysis suggests that SIVmac had already emerged by the early 1970s and had begun to diverge into distinct lineages. Furthermore, our findings suggest that pathogenic SIV strains may have been more widely distributed than previously appreciated, raising the possibility that additional isolates may await discovery.

Tracking interspecies transmission and long-term evolution of an ancient retrovirus using the genomes of modern mammals.

Mammalian genomes typically contain hundreds of thousands of endogenous retroviruses (ERVs), derived from ancient retroviral infections. Using this molecular 'fossil' record, we reconstructed the natural history of a specific retrovirus lineage (ERV-Fc) that disseminated widely between ~33 and ~15 million years ago, corresponding to the Oligocene and early Miocene epochs. Intercontinental viral spread, numerous instances of interspecies transmission and emergence in hosts representing at least 11 mammalian orders, and a significant role for recombination in diversification of this viral lineage were also revealed. By reconstructing the canonical retroviral genes, we identified patterns of adaptation consistent with selection to maintain essential viral protein functions. Our results demonstrate the unique potential of the ERV fossil record for studying the processes of viral spread and emergence as they play out across macro-evolutionary timescales, such that looking back in time may prove insightful for predicting the long-term consequences of newly emerging viral infections.

Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif.

APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

Endogenous Retroviruses in the Genomics Era.

Endogenous retroviruses comprise millions of discrete genetic loci distributed within the genomes of extant vertebrates. These sequences, which are clearly related to exogenous retroviruses, represent retroviral infections of the deep past, and their abundance suggests that retroviruses were a near-constant presence throughout the evolutionary history of modern vertebrates. Endogenous retroviruses contribute in myriad ways to the evolution of host genomes, as mutagens and as sources of genetic novelty (both coding and regulatory) to be acted upon by the twin engines of random genetic drift and natural selection. Importantly, the richness and complexity of endogenous retrovirus data can be used to understand how viruses spread and adapt on evolutionary timescales by combining population genetics and evolutionary theory with a detailed understanding of retrovirus biology (gleaned from the study of extant retroviruses). In addition to revealing the impact of viruses on organismal evolution, such studies can help us better understand, by looking back in time, how life-history traits, as well as ecological and geological events, influence the movement of viruses within and between populations.

Multimodality vaccination against clade C SHIV: partial protection against mucosal challenges with a heterologous tier 2 virus.

We sought to test whether vaccine-induced immune responses could protect rhesus macaques (RMs) against upfront heterologous challenges with an R5 simian-human immunodeficiency virus, SHIV-2873Nip. This SHIV strain exhibits many properties of transmitted HIV-1, such as tier 2 phenotype (relatively difficult to neutralize), exclusive CCR5 tropism, and gradual disease progression in infected RMs. Since no human AIDS vaccine recipient is likely to encounter an HIV-1 strain that exactly matches the immunogens, we immunized the RMs with recombinant Env proteins heterologous to the challenge virus. For induction of immune responses against Gag, Tat, and Nef, we explored a strategy of immunization with overlapping synthetic peptides (OSP). The immune responses against Gag and Tat were finally boosted with recombinant proteins. The vaccinees and a group of ten control animals were given five low-dose intrarectal (i.r.) challenges with SHIV-2873Nip. All controls and seven out of eight vaccinees became systemically infected; there was no significant difference in viremia levels of vaccinees vs. controls. Prevention of viremia was observed in one vaccinee which showed strong boosting of virus-specific cellular immunity during virus exposures. The protected animal showed no challenge virus-specific neutralizing antibodies in the TZM-bl or A3R5 cell-based assays and had low-level ADCC activity after the virus exposures. Microarray data strongly supported a role for cellular immunity in the protected animal. Our study represents a case of protection against heterologous tier 2 SHIV-C by vaccine-induced, virus-specific cellular immune responses.

A novel recombinant retrovirus in the genomes of modern birds combines features of avian and mammalian retroviruses.

UNLABELLED: Endogenous retroviruses (ERVs) represent ancestral sequences of modern retroviruses or their extinct relatives. The majority of ERVs cluster alongside exogenous retroviruses into two main groups based on phylogenetic analyses of the reverse transcriptase (RT) enzyme. Class I includes gammaretroviruses, and class II includes lentiviruses and alpha-, beta-, and deltaretroviruses. However, analyses of the transmembrane subunit (TM) of the envelope glycoprotein (env) gene result in a different topology for some retroviruses, suggesting recombination events in which heterologous env sequences have been acquired. We previously demonstrated that the TM sequences of five of the six genera of orthoretroviruses can be divided into three types, each of which infects a distinct set of vertebrate classes. Moreover, these classes do not always overlap the host range of the associated RT classes. Thus, recombination resulting in acquisition of a heterologous env gene could in theory facilitate cross-species transmissions across vertebrate classes, for example, from mammals to reptiles. Here we characterized a family of class II avian ERVs, "TgERV-F," that acquired a mammalian gammaretroviral env sequence. Although TgERV-F clusters near a sister clade to alpharetroviruses, its genome also has some features of betaretroviruses. We offer evidence that this unusual recombinant has circulated among several avian orders and may still have infectious members. In addition to documenting the infection of a nongalliform avian species by a mammalian retrovirus, TgERV-F also underscores the importance of env sequences in reconstructing phylogenies and supports a possible role for env swapping in allowing cross-species transmissions across wide taxonomic distances. IMPORTANCE: Retroviruses can sometimes acquire an envelope gene (env) from a distantly related retrovirus. Since env is a key determinant of host range, such an event affects the host range of the recombinant virus and can lead to the creation of novel retroviral lineages. Retroviruses insert viral DNA into the host DNA during infection, and therefore vertebrate genomes contain a "fossil record" of endogenous retroviral sequences thought to represent past infections of germ cells. We examined endogenous retroviral sequences in avian genomes for evidence of recombination events involving env. Although cross-species transmissions of retroviruses between vertebrate classes (from mammals to birds, for example) are thought to be rare, we here characterized a group of avian retroviruses that acquired an env sequence from a mammalian retrovirus. We offer evidence that this unusual recombinant circulated among songbirds 2 to 4 million years ago and has remained active into the recent past.

Pushing the endogenous envelope.

The majority of retroviral envelope glycoproteins characterized to date are typical of type I viral fusion proteins, having a receptor binding subunit associated with a fusion subunit. The fusion subunits of lentiviruses and alpha-, beta-, delta- and gammaretroviruses have a very conserved domain organization and conserved features of secondary structure, making them suitable for phylogenetic analyses. Such analyses, along with sequence comparisons, reveal evidence of numerous recombination events in which retroviruses have acquired envelope glycoproteins from heterologous sequences. Thus, the envelope gene (env) can have a history separate from that of the polymerase gene (pol), which is the most commonly used gene in phylogenetic analyses of retroviruses. Focusing on the fusion subunits of the genera listed above, we describe three distinct types of retroviral envelope glycoproteins, which we refer to as gamma-type, avian gamma-type and beta-type. By tracing these types within the 'fossil record' provided by endogenous retroviruses, we show that they have surprisingly distinct evolutionary histories and dynamics, with important implications for cross-species transmissions and the generation of novel lineages. These findings validate the utility of env sequences in contributing phylogenetic signal that enlarges our understanding of retrovirus evolution.

Gain-of-sensitivity mutations in a Trim5-resistant primary isolate of pathogenic SIV identify two independent conserved determinants of Trim5α specificity.

Retroviral capsid recognition by Trim5 blocks productive infection. Rhesus macaques harbor three functionally distinct Trim5 alleles: Trim5α(Q) , Trim5α(TFP) and Trim5(CypA) . Despite the high degree of amino acid identity between Trim5α(Q) and Trim5α(TFP) alleles, the Q/TFP polymorphism results in the differential restriction of some primate lentiviruses, suggesting these alleles differ in how they engage these capsids. Simian immunodeficiency virus of rhesus macaques (SIVmac) evolved to resist all three alleles. Thus, SIVmac provides a unique opportunity to study a virus in the context of the Trim5 repertoire that drove its evolution in vivo. We exploited the evolved rhesus Trim5α resistance of this capsid to identify gain-of-sensitivity mutations that distinguish targets between the Trim5α(Q) and Trim5α(TFP) alleles. While both alleles recognize the capsid surface, Trim5α(Q) and Trim5α(TFP) alleles differed in their ability to restrict a panel of capsid chimeras and single amino acid substitutions. When mapped onto the structure of the SIVmac239 capsid N-terminal domain, single amino acid substitutions affecting both alleles mapped to the ß-hairpin. Given that none of the substitutions affected Trim5α(Q) alone, and the fact that the ß-hairpin is conserved among retroviral capsids, we propose that the ß-hairpin is a molecular pattern widely exploited by Trim5α proteins. Mutations specifically affecting rhesus Trim5α(TFP) (without affecting Trim5α(Q) ) surround a site of conservation unique to primate lentiviruses, overlapping the CPSF6 binding site. We believe targeting this site is an evolutionary innovation driven specifically by the emergence of primate lentiviruses in Africa during the last 12 million years. This modularity in targeting may be a general feature of Trim5 evolution, permitting different regions of the PRYSPRY domain to evolve independent interactions with capsid.

Susceptibility to repeated, low-dose, rectal SHIVSF162P3 challenge is independent of TRIM5 genotype in rhesus macaques.

Infections following repeated, low-dose (RLD), mucal S(H)IV exposures of macaques are used to model sexual HIV exposures for biomedical prevention testing. Different susceptibilities among animals can complicate study designs. In rhesus macaques, TRIM5 alleles Q, CypA, and TFP are resistance factors for infection with some S(H)IV strains, but not for SIVmac239 due to its capsid properties. SIVmac239-derived SHIVSF162P3 has been demonstrated to reproducibly infect mucosally in vaginal and rectal RLD models. To further test the suitability of SHIVSF162P3 for RLD models, we studied the influence of the TRIM5 genotype on susceptibility to rectal RLD infection and on plasma viremia by analyzing 43 male Indian rhesus macaques from control arms of completed studies. The median number of exposures required for infection was three (Q/Q, n=4) (TRIM5 alleles, number of macaques, respectively), four (Q/CypA, n=7), three (TFP/Q, n=15), three (TFP/TFP, n=15), and two (TFP/CypA, n=2); TRIM5(CypA/CypA) was not represented in our study. Median peak viremia (log10 viral copies/ml) in infected animals was 7.4 (Q/Q, n=4), 7.2 (Q/CypA, n=6), 7.3 (TFP/Q, n=13), 7.1 (TFP/TFP, n=15), and 6.5 (TFP/CypA; n=2). Neither susceptibility nor peak viremia was significantly different (log rank test, Kruskal-Wallis test, respectively). Rhesus macaques' susceptibility to RLD SHIVSF162P3 is independent of the TRIM5 TFP, CypA, and Q alleles, with the limitation that the power to detect any impact of CypA/CypA and TFP/CypA genotypes was nonexistent or low, due to absence or infrequency, respectively. The finding that TRIM5 alleles do not restrict mucosal infection or ensuing replication rates suggests that SHIVSF162P3 is indeed suitable for RLD experimentation.

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges.

We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8⁺ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4ß7-expressing CD4⁺ T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection.