Reverse transcriptase inhibitors prevent liver abscess formation during Escherichia coli bloodstream infection.

The presence of bacteria in the bloodstream is associated with severe clinical outcomes. In mice, intravenous inoculation of Escherichia coli can lead to the formation of macroscopic abscesses in the liver. Abscesses are regions of severe necrosis and consist of millions of bacteria surrounded by inflammatory immune cells. Liver abscess susceptibility varies widely across strains of mice, but the host factors governing this variation are unknown. Here, we profiled hepatic transcriptomes in mice with varying susceptibility to liver abscess formation. We found that transcripts from endogenous retroviruses (ERVs) are robustly induced in the liver by E. coli infection and ERV expression positively correlates with the frequency of abscess formation. Hypothesizing that ERV-encoded reverse transcriptase may generate cytoplasmic DNA and heighten inflammatory responses, we tested whether nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) influence abscess formation. Strikingly, a single NRTI dose administered immediately following E. coli inoculation prevented abscess formation, leading to a concomitant 100,000-fold reduction in bacterial burden. We provide evidence that NRTIs inhibit abscess formation by preventing the tissue necrosis that facilitates bacterial replication. Together, our findings suggest that endogenous reverse transcriptases drive inflammatory responses during bacterial bloodstream infection to drive abscess formation. The high efficacy of NRTIs in preventing abscess formation suggests that the consequences of reverse transcription on inflammation should be further examined, particularly in infectious diseases where inflammation drives negative clinical outcomes, such as sepsis.

Conserved residues of the immunosuppressive domain of MLV are essential for regulating the fusion-critical SU-TM disulfide bond.

The Env protein of murine leukemia virus (MLV) is the prototype of a large clade of retroviral fusogens, collectively known as gamma-type Envs. Gamma-type Envs are found in retroviruses and endogenous retroviruses (ERVs) representing a broad range of vertebrate hosts. All gamma-type Envs contain a highly conserved stretch of 26-residues in the transmembrane subunit (TM) comprising two motifs, a putative immunosuppressive domain (ISD) and a CX6CC motif. Extraordinary conservation of the ISD and its invariant association with the CX6CC suggests a fundamental contribution to Env function. To investigate ISD function, we characterized several mutants with single amino acid substitutions at conserved positions in the MLV ISD. A majority abolished infectivity, although we did not observe a corresponding loss in intrinsic ability to mediate membrane fusion. Ratios of the surface subunit (SU) to capsid protein (CA) in virions were diminished for a majority of the ISD mutants, while TM:CA ratios were similar to wild type. Specific loss of SU reflected premature isomerization of the labile disulfide bond that links SU and TM prior to fusion. Indeed, all non-infectious mutants displayed significantly lower disulfide stability than wild-type Env. These results reveal a role for ISD positions 2, 3, 4, 7, and 10 in regulating a late step in entry after fusion peptide insertion but prior to creation of the fusion pore. This implies that the ISD is part of a larger domain, comprising the ISD and CX6CC motifs, that is critical for the formation and regulation of the metastable, intersubunit disulfide bond.IMPORTANCEThe gamma-type Env is a prevalent viral fusogen, found within retroviruses and endogenous retroviruses across vertebrate species and in filoviruses such as Ebolavirus. The fusion mechanism of gamma-type Envs is unique from other Class I fusogens such as those of influenza A virus and HIV-1. Gamma-type Envs contain a hallmark feature known as the immunosuppressive domain (ISD) that has been the subject of some controversy in the literature surrounding its putative immunosuppressive effects. Despite the distinctive conservation of the ISD, little has been done to investigate the role of this region for the function of this widespread fusogen. Our work demonstrates the importance of the ISD for the function of gamma-type Envs in infection, particularly in regulating the intermediate steps of membrane fusion. Understanding the fusion mechanism of gamma-type Envs has broad implications for understanding the entry of extant viruses and aspects of host biology connected to co-opted endogenous gamma-type Envs.

APOBEC3F Constitutes a Barrier to Successful Cross-Species Transmission of Simian Immunodeficiency Virus SIVsmm to Humans.

Simian immunodeficiency virus infecting sooty mangabeys (SIVsmm) has been transmitted to humans on at least nine occasions, giving rise to human immunodeficiency virus type 2 (HIV-2) groups A to I. SIVsmm isolates replicate in human T cells and seem capable of overcoming major human restriction factors without adaptation. However, only groups A and B are responsible for the HIV-2 epidemic in sub-Saharan Africa, and it is largely unclear whether adaptive changes were associated with spread in humans. To address this, we examined the sensitivity of infectious molecular clones (IMCs) of five HIV-2 strains and representatives of five different SIVsmm lineages to various APOBEC3 proteins. We confirmed that SIVsmm strains replicate in human T cells, albeit with more variable replication fitness and frequently lower efficiency than HIV-2 IMCs. Efficient viral propagation was generally dependent on intact vif genes, highlighting the need for counteraction of APOBEC3 proteins. On average, SIVsmm was more susceptible to inhibition by human APOBEC3D, -F, -G, and -H than HIV-2. For example, human APOBEC3F reduced infectious virus yield of SIVsmm by ∼80% but achieved only ∼40% reduction in the case of HIV-2. Functional and mutational analyses of human- and monkey-derived alleles revealed that an R128T polymorphism in APOBEC3F contributes to species-specific counteraction by HIV-2 and SIVsmm Vifs. In addition, a T84S substitution in SIVsmm Vif increased its ability to counteract human APOBEC3F. Altogether, our results confirm that SIVsmm Vif proteins show intrinsic activity against human APOBEC3 proteins but also demonstrate that epidemic HIV-2 strains evolved an increased ability to counteract this class of restriction factors during human adaptation. IMPORTANCE Viral zoonoses pose a significant threat to human health, and it is important to understand determining factors. SIVs infecting great apes gave rise to HIV-1. In contrast, SIVs infecting African monkey species have not been detected in humans, with one notable exception. SIVsmm from sooty mangabeys has crossed the species barrier to humans on at least nine independent occasions and seems capable of overcoming many innate defense mechanisms without adaptation. Here, we confirmed that SIVsmm Vif proteins show significant activity against human APOBEC3 proteins. Our analyses also revealed, however, that different lineages of SIVsmm are significantly more susceptible to inhibition by various human APOBEC3 proteins than HIV-2 strains. Mutational analyses suggest that an R128T substitution in APOBEC3F and a T84S change in Vif contribute to species-specific counteraction by HIV-2 and SIVsmm. Altogether, our results support that epidemic HIV-2 strains acquired increased activity against human APOBEC3 proteins to clear this restrictive barrier.

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.

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.

Nomenclature for endogenous retrovirus (ERV) loci.

Retroviral integration into germline DNA can result in the formation of a vertically inherited proviral sequence called an endogenous retrovirus (ERV). Over the course of their evolution, vertebrate genomes have accumulated many thousands of ERV loci. These sequences provide useful retrospective information about ancient retroviruses, and have also played an important role in shaping the evolution of vertebrate genomes. There is an immediate need for a unified system of nomenclature for ERV loci, not only to assist genome annotation, but also to facilitate research on ERVs and their impact on genome biology and evolution. In this review, we examine how ERV nomenclatures have developed, and consider the possibilities for the implementation of a systematic approach for naming ERV loci. We propose that such a nomenclature should not only provide unique identifiers for individual loci, but also denote orthologous relationships between ERVs in different species. In addition, we propose that-where possible-mnemonic links to previous, well-established names for ERV loci and groups should be retained. We show how this approach can be applied and integrated into existing taxonomic and nomenclature schemes for retroviruses, ERVs and transposable elements.

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.

TRIM5α Resistance Escape Mutations in the Capsid Are Transferable between Simian Immunodeficiency Virus Strains.

TRIM5α polymorphism limits and complicates the use of simian immunodeficiency virus (SIV) for evaluation of human immunodeficiency virus (HIV) vaccine strategies in rhesus macaques. We previously reported that the TRIM5α-sensitive SIV from sooty mangabeys (SIVsm) clone SIVsmE543-3 acquired amino acid substitutions in the capsid that overcame TRIM5α restriction when it was passaged in rhesus macaques expressing restrictive TRIM5α alleles. Here we generated TRIM5α-resistant clones of the related SIVsmE660 strain without animal passage by introducing the same amino acid capsid substitutions. We evaluated one of the variants in rhesus macaques expressing permissive and restrictive TRIM5α alleles. The SIVsmE660 variant infected and replicated in macaques with restrictive TRIM5α genotypes as efficiently as in macaques with permissive TRIM5α genotypes. These results demonstrated that mutations in the SIV capsid can confer SIV resistance to TRIM5α restriction without animal passage, suggesting an applicable method to generate more diverse SIV strains for HIV vaccine studies. IMPORTANCE: Many strains of SIV from sooty mangabey monkeys are susceptible to resistance by common rhesus macaque TRIM5α alleles and result in reduced virus acquisition and replication in macaques that express these restrictive alleles. We previously observed that spontaneous variations in the capsid gene were associated with improved replication in macaques, and the introduction of two amino acid changes in the capsid transfers this improved replication to the parent clone. In the present study, we introduced these mutations into a related but distinct strain of SIV that is commonly used for challenge studies for vaccine trials. These mutations also improved the replication of this strain in macaques with the restrictive TRIM5α genotype and thus will eliminate the confounding effects of TRIM5α in vaccine studies.

Evolutionary and Functional Analysis of Old World Primate TRIM5 Reveals the Ancient Emergence of Primate Lentiviruses and Convergent Evolution Targeting a Conserved Capsid Interface.

The widespread distribution of lentiviruses among African primates, and the lack of severe pathogenesis in many of these natural reservoirs, are taken as evidence for long-term co-evolution between the simian immunodeficiency viruses (SIVs) and their primate hosts. Evidence for positive selection acting on antiviral restriction factors is consistent with virus-host interactions spanning millions of years of primate evolution. However, many restriction mechanisms are not virus-specific, and selection cannot be unambiguously attributed to any one type of virus. We hypothesized that the restriction factor TRIM5, because of its unique specificity for retrovirus capsids, should accumulate adaptive changes in a virus-specific fashion, and therefore, that phylogenetic reconstruction of TRIM5 evolution in African primates should reveal selection by lentiviruses closely related to modern SIVs. We analyzed complete TRIM5 coding sequences of 22 Old World primates and identified a tightly-spaced cluster of branch-specific adaptions appearing in the Cercopithecinae lineage after divergence from the Colobinae around 16 million years ago. Functional assays of both extant TRIM5 orthologs and reconstructed ancestral TRIM5 proteins revealed that this cluster of adaptations in TRIM5 specifically resulted in the ability to restrict Cercopithecine lentiviruses, but had no effect (positive or negative) on restriction of other retroviruses, including lentiviruses of non-Cercopithecine primates. The correlation between lineage-specific adaptations and ability to restrict viruses endemic to the same hosts supports the hypothesis that lentiviruses closely related to modern SIVs were present in Africa and infecting the ancestors of Cercopithecine primates as far back as 16 million years ago, and provides insight into the evolution of TRIM5 specificity.

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.

APOBEC3G polymorphism as a selective barrier to cross-species transmission and emergence of pathogenic SIV and AIDS in a primate host.

Cellular restriction factors, which render cells intrinsically resistant to viruses, potentially impose genetic barriers to cross-species transmission and emergence of viral pathogens in nature. One such factor is APOBEC3G. To overcome APOBEC3G-mediated restriction, many lentiviruses encode Vif, a protein that targets APOBEC3G for degradation. As with many restriction factor genes, primate APOBEC3G displays strong signatures of positive selection. This is interpreted as evidence that the primate APOBEC3G locus reflects a long-term evolutionary "arms-race" between retroviruses and their primate hosts. Here, we provide direct evidence that APOBEC3G has functioned as a barrier to cross-species transmission, selecting for viral resistance during emergence of the AIDS-causing pathogen SIVmac in captive colonies of Asian macaques in the 1970s. Specifically, we found that rhesus macaques have multiple, functionally distinct APOBEC3G alleles, and that emergence of SIVmac and simian AIDS required adaptation of the virus to evade APOBEC3G-mediated restriction. Our evidence includes the first comparative analysis of APOBEC3G polymorphism and function in both a reservoir and recipient host species (sooty mangabeys and rhesus macaques, respectively), and identification of adaptations unique to Vif proteins of the SIVmac lineage that specifically antagonize rhesus APOBEC3G alleles. By demonstrating that interspecies variation in a known restriction factor selected for viral counter-adaptations in the context of a documented case of cross-species transmission, our results lend strong support to the evolutionary "arms-race" hypothesis. Importantly, our study confirms that APOBEC3G divergence can be a critical determinant of interspecies transmission and emergence of primate lentiviruses, including viruses with the potential to infect and spread in human populations.

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.

Passive immunization of macaques with polyclonal anti-SHIV IgG against a heterologous tier 2 SHIV: outcome depends on IgG dose.

BACKGROUND: A key goal for HIV-1 envelope immunogen design is the induction of cross-reactive neutralizing antibodies (nAbs). As AIDS vaccine recipients will not be exposed to strains exactly matching any immunogens due to multiple HIV-1 quasispecies circulating in the human population worldwide, heterologous SHIV challenges are essential for realistic vaccine efficacy testing in primates. We assessed whether polyclonal IgG, isolated from rhesus monkeys (RMs) with high-titer nAbs (termed SHIVIG), could protect RMs against the R5-tropic tier-2 SHIV-2873Nip, which was heterologous to the viruses or HIV-1 envelopes that had elicited SHIVIG. RESULTS: SHIVIG demonstrated binding to HIV Gag, Tat, and Env of different clades and competed with the broadly neutralizing antibodies b12, VRC01, 4E10, and 17b. SHIVIG neutralized tier 1 and tier 2 viruses, including SHIV-2873Nip. NK-cell depletion decreased the neutralizing activity of SHIVIG 20-fold in PBMC assays. Although SHIVIG neutralized SHIV-2873Nip in vitro, this polyclonal IgG preparation failed to prevent acquisition after repeated intrarectal low-dose virus challenges, but at a dose of 400 mg/kg, it significantly lowered peak viremia (P = 0.001). Unexpectedly, single-genome analysis revealed a higher number of transmitted variants at the low dose of 25 mg/kg, implying increased acquisition at low SHIVIG levels. In vitro, SHIVIG demonstrated complement-mediated Ab-dependent enhancement of infection (C'-ADE) at concentrations similar to those observed in plasmas of RMs treated with 25 mg/kg of SHIVIG. CONCLUSION: Our primate model data suggest a dual role for polyclonal anti-HIV-1 Abs depending on plasma levels upon virus encounter.

Rapid adversarial co-evolution of viruses and cellular restriction factors.

Since the discovery of viruses over a century ago, virologists have recognized that host genetics plays a major role in viral tropism and the distribution of viruses in nature. Traditionally, studies of tropism have centered on identification of cellular factors required for viral replication, such as cell-surface entry receptors. However, over the past 20 years, there has been a steady increase in the identification and characterization of restriction factors (RFs), here defined as dominant cellular factors that have evolved specifically to interfere with viral replication. Genetic studies suggest that restriction factors impose significant barriers to interspecies movement of viruses and are therefore critical determinants of viral tropism. Furthermore, the scope of the ever-expanding list of restriction factors, and the variety of antiviral mechanisms they represent, testifies to the extraordinary impact viruses have had on organismal evolution-an impact hitherto underappreciated by evolutionary biologists and virologists alike. Recent studies of RF-encoding genes that combine molecular evolutionary analysis with functional assays illustrate the potential for asking questions about virus-host interactions as they play out in natural populations and across evolutionary timescales. Most notably, it has become common to apply tests of positive selection to RF genes and couple these analyses with virological assays, to reveal evidence for antagonistic virus-host co-evolution. Herein, I summarize recent work on the evolutionary genetics of mammalian RFs, particularly those of humans, non-human primates, and model organisms, and how RFs can reveal the influence of virus-host interactions on organismal evolution. Because intensive investigation of RF evolution is fairly new (and because there is still much to learn), the discussion is organized around five broad, outstanding questions that will need to be answered before we can fully appreciate the evolutionary biology of restriction.

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.

Predicting potential SARS-CoV-2 mutations of concern via full quantum mechanical modelling.

Ab initio quantum mechanical models can characterize and predict intermolecular binding, but only recently have models including more than a few hundred atoms gained traction. Here, we simulate the electronic structure for approximately 13 000 atoms to predict and characterize binding of SARS-CoV-2 spike variants to the human ACE2 (hACE2) receptor using the quantum mechanics complexity reduction (QM-CR) approach. We compare four spike variants in our analysis: Wuhan, Omicron, and two Omicron-based variants. To assess binding, we mechanistically characterize the energetic contribution of each amino acid involved, and predict the effect of select single amino acid mutations. We validate our computational predictions experimentally by comparing the efficacy of spike variants binding to cells expressing hACE2. At the time we performed our simulations (December 2021), the mutation A484K which our model predicted to be highly beneficial to ACE2 binding had not been identified in epidemiological surveys; only recently (August 2023) has it appeared in variant BA.2.86. We argue that our computational model, QM-CR, can identify mutations critical for intermolecular interactions and inform the engineering of high-specificity interactors.

Binding of anti-membrane-proximal gp41 monoclonal antibodies to CD4-liganded and -unliganded human immunodeficiency virus type 1 and simian immunodeficiency virus virions.

The broadly neutralizing monoclonal antibodies (MAbs) 4E10, 2F5, and Z13e1 target membrane-proximal external region (MPER) epitopes of HIV-1 gp41 in a manner that remains controversial. The requirements for initial lipid bilayer binding and/or CD4 ligation have been proposed. To further investigate these issues, we probed for binding of these MAbs to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions with protein A-conjugated gold (PAG) nanoparticles using negative-stain electron microscopy. We found moderate levels of PAG associated with unliganded HIV-1 and SIV virions incubated with the three MAbs. Significantly higher levels of PAG were associated with CD4-liganded HIV-1 (epitope-positive) but not SIV (epitope-negative) virions. A chimeric SIV virion displaying the HIV-1 4E10 epitope also showed significantly higher PAG association after CD4 ligation and incubation with 4E10. MAbs accumulated rapidly on CD4-liganded virions and slowly on unliganded virions, although both reached similar levels in time. Anti-MPER epitope-specific binding was stable to washout. Virions incubated with an irrelevant MAb or CD4-only (no MAb) showed negligible PAG association, as did a vesicle-rich fraction devoid of virions. Preincubation with Fab 4E10 inhibited both specific and nonspecific 4E10 IgG binding. Our data provide evidence for moderate association of anti-MPER MAbs to viral surfaces but not lipid vesicles, even in the absence of cognate epitopes. Significantly greater MAb interaction occurs in epitope-positive virions following long incubation or CD4 ligation. These findings are consistent with a two-stage binding model where these anti-MPER MAbs bind first to the viral lipid bilayer and then to the MPER epitopes following spontaneous or induced exposure.

TRIM5 suppresses cross-species transmission of a primate immunodeficiency virus and selects for emergence of resistant variants in the new species.

Simian immunodeficiency viruses of sooty mangabeys (SIVsm) are the source of multiple, successful cross-species transmissions, having given rise to HIV-2 in humans, SIVmac in rhesus macaques, and SIVstm in stump-tailed macaques. Cellular assays and phylogenetic comparisons indirectly support a role for TRIM5alpha, the product of the TRIM5 gene, in suppressing interspecies transmission and emergence of retroviruses in nature. Here, we investigate the in vivo role of TRIM5 directly, focusing on transmission of primate immunodeficiency viruses between outbred primate hosts. Specifically, we retrospectively analyzed experimental cross-species transmission of SIVsm in two cohorts of rhesus macaques and found a significant effect of TRIM5 genotype on viral replication levels. The effect was especially pronounced in a cohort of animals infected with SIVsmE543-3, where TRIM5 genotype correlated with approximately 100-fold to 1,000-fold differences in viral replication levels. Surprisingly, transmission occurred even in individuals bearing restrictive TRIM5 genotypes, resulting in attenuation of replication rather than an outright block to infection. In cell-culture assays, the same TRIM5 alleles associated with viral suppression in vivo blocked infectivity of two SIVsm strains, but not the macaque-adapted strain SIVmac239. Adaptations appeared in the viral capsid in animals with restrictive TRIM5 genotypes, and similar adaptations coincide with emergence of SIVmac in captive macaques in the 1970s. Thus, host TRIM5 can suppress viral replication in vivo, exerting selective pressure during the initial stages of cross-species transmission.

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.

Significant protection against high-dose simian immunodeficiency virus challenge conferred by a new prime-boost vaccine regimen.

We constructed vaccine vectors based on live recombinant vesicular stomatitis virus (VSV) and a Semliki Forest virus (SFV) replicon (SFVG) that propagates through expression of the VSV glycoprotein (G). These vectors expressing simian immunodeficiency virus (SIV) Gag and Env proteins were used to vaccinate rhesus macaques with a new heterologous prime-boost regimen designed to optimize induction of antibody. Six vaccinated animals and six controls were then given a high-dose mucosal challenge with the diverse SIVsmE660 quasispecies. All control animals became infected and had peak viral RNA loads of 10(6) to 10(8) copies/ml. In contrast, four of the vaccinees showed significant (P = 0.03) apparent sterilizing immunity and no detectable viral loads. Subsequent CD8(+) T cell depletion confirmed the absence of SIV infection in these animals. The two other vaccinees had peak viral loads of 7 × 10(5) and 8 × 10(3) copies/ml, levels below those of all of the controls, and showed undetectable virus loads by day 42 postchallenge. The vaccine regimen induced high-titer prechallenge serum neutralizing antibodies (nAbs) to some cloned SIVsmE660 Env proteins, but antibodies able to neutralize the challenge virus swarm were not detected. The cellular immune responses induced by the vaccine were generally weak and did not correlate with protection. Although the immune correlates of protection are not yet clear, the heterologous VSV/SFVG prime-boost is clearly a potent vaccine regimen for inducing virus nAbs and protection against a heterogeneous viral swarm.