Co-option of an endogenous retrovirus envelope for host defense in hominid ancestors

Endogenous retroviral sequences provide a molecular fossil record of ancient infections whose analysis might illuminate mechanisms of viral extinction. A close relative of gammaretroviruses, HERV-T, circulated in primates for ~25 million years (MY) before apparent extinction within the past ~8 MY. Construction of a near-complete catalog of HERV-T fossils in primate genomes allowed us to estimate a ~32 MY old ancestral sequence and reconstruct a functional envelope protein (ancHTenv) that could support infection of a pseudotyped modern gammaretrovirus. Using ancHTenv, we identify monocarboxylate transporter-1 (MCT-1) as a receptor used by HERV-T for attachment and infection. A single HERV-T provirus in hominid genomes includes an env gene (hsaHTenv) that has been uniquely preserved. This apparently exapted HERV-T env could not support virion infection but could block ancHTenv mediated infection, by causing MCT-1 depletion from cell surfaces. Thus, hsaHTenv may have contributed to HERV-T extinction, and could also potentially regulate cellular metabolism

The RNA binding specificity of human APOBEC3 proteins resembles that of HIV-1 nucleocapsid

The APOBEC3 (A3) cytidine deaminases are antiretroviral proteins, whose targets include human immunodeficiency virus type-1 (HIV-1). Their incorporation into viral particles is critical for antiviral activity and is driven by interactions with the RNA molecules that are packaged into virions. However, it is unclear whether A3 proteins preferentially target RNA molecules that are destined to be packaged and if so, how. Using cross-linking immunoprecipitation sequencing (CLIP-seq), we determined the RNA binding preferences of the A3F, A3G and A3H proteins. We found that A3 proteins bind preferentially to RNA segments with particular properties, both in cells and in virions. Specifically, A3 proteins target RNA sequences that are G-rich and/or A-rich and are not scanned by ribosomes during translation. Comparative analyses of HIV-1 Gag, nucleocapsid (NC) and A3 RNA binding to HIV-1 RNA in cells and virions revealed the striking finding that A3 proteins partially mimic the RNA binding specificity of the HIV-1 NC protein. These findings suggest a model for A3 incorporation into HIV-1 virions in which an NC-like RNA binding specificity is determined by nucleotide composition rather than sequence. This model reconciles the promiscuity of A3 RNA binding that has been observed in previous studies with a presumed advantage that would accompany selective binding to RNAs that are destined to be packaged into virions

Species-Specific Activity of HIV-1 Vpu and Positive Selection of Tetherin Transmembrane Domain Variants

Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh), African green monkeys (agm) and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins

Interferon-stimulated gene (ISG)-expression screening reveals the specific antibunyaviral activity of ISG20

Bunyaviruses pose a significant threat to human health, prosperity and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon stimulated genes (ISGs) whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional ribonuclease activity. Through use of an infectious VLP assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taken together, we report that ISG20 is a broad and potent antibunyaviral factor yet some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance could influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance

Reconstitution of an Infectious Human Endogenous Retrovirus

The human genome represents a fossil record of ancient retroviruses that once replicated in the ancestors of contemporary humans. Indeed, approximately 8% of human DNA is composed of sequences that are recognizably retroviral. Despite occasional reports associating human endogenous retrovirus (HERV) expression with human disease, almost all HERV genomes contain obviously inactivating mutations, and none are thought to be capable of replication. Nonetheless, one family of HERVs, namely HERV-K(HML-2), may have replicated in human ancestors less than 1 million years ago. By deriving a consensus sequence, we reconstructed a proviral clone (HERV-K(CON)) that likely resembles the progenitor of HERV-K(HML-2) variants that entered the human genome within the last few million years. We show that HERV-K(CON) Gag and protease proteins mediate efficient assembly and processing into retrovirus-like particles. Moreover, reporter genes inserted into the HERV-K(CON) genome and packaged into HERV-K particles are capable of infectious transfer and stable integration in a manner that requires reverse transcription. Additionally, we show that HERV-K(CON) Env is capable of pseudotyping HIV-1 particles and mediating entry into human and nonhuman cell lines. Furthermore, we show that HERV-K(CON) is resistant to inhibition by the human retrovirus restriction factors tripartite motif 5α and apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) 3G but is inhibited by APOBEC 3F. Overall, the resurrection of this extinct infectious agent in a functional form from molecular fossils should enable studies of the molecular virology and pathogenic potential of this ancient human retrovirus

Assisted evolution enables HIV-1 to overcome a high trim5α-imposed genetic barrier to rhesus macaque tropism

Diversification of antiretroviral factors during host evolution has erected formidable barriers to cross-species retrovirus transmission. This phenomenon likely protects humans from infection by many modern retroviruses, but it has also impaired the development of primate models of HIV-1 infection. Indeed, rhesus macaques are resistant to HIV-1, in part due to restriction imposed by the TRIM5α protein (rhTRIM5α). Initially, we attempted to derive rhTRIM5α-resistant HIV-1 strains using two strategies. First, HIV-1 was passaged in engineered human cells expressing rhTRIM5α. Second, a library of randomly mutagenized capsid protein (CA) sequences was screened for mutations that reduced rhTRIM5α sensitivity. Both approaches identified several individual mutations in CA that reduced rhTRIM5α sensitivity. However, neither approach yielded mutants that were fully resistant, perhaps because the locations of the mutations suggested that TRIM5α recognizes multiple determinants on the capsid surface. Moreover, even though additive effects of various CA mutations on HIV-1 resistance to rhTRIM5α were observed, combinations that gave full resistance were highly detrimental to fitness. Therefore, we employed an 'assisted evolution' approach in which individual CA mutations that reduced rhTRIM5α sensitivity without fitness penalties were randomly assorted in a library of viral clones containing synthetic CA sequences. Subsequent passage of the viral library in rhTRIM5α-expressing cells resulted in the selection of individual viral species that were fully fit and resistant to rhTRIM5α. These viruses encoded combinations of five mutations in CA that conferred complete or near complete resistance to the disruptive effects of rhTRIM5α on incoming viral cores, by abolishing recognition of the viral capsid. Importantly, HIV-1 variants encoding these CA substitutions and SIVmac239 Vif replicated efficiently in primary rhesus macaque lymphocytes. These findings demonstrate that rhTRIM5α is difficult to but not impossible to evade, and doing so should facilitate the development of primate models of HIV-1 infection

Natural Variation in Vif: Differential Impact on APOBEC3G/3F and a Potential Role in HIV-1 Diversification

The HIV-1 Vif protein counteracts the antiviral activity exhibited by the host cytidine deaminases APOBEC3G and APOBEC3F. Here, we show that defective vif alleles can readily be found in HIV-1 isolates and infected patients. Single residue changes in the Vif protein sequence are sufficient to cause the loss of Vif-induced APOBEC3 neutralization. Interestingly, not all the detected defects lead to a complete inactivation of Vif function since some mutants retained selective neutralizing activity against APOBEC3F but not APOBEC3G or vice versa. Concordantly, independently hypermutated proviruses with distinguishable patterns of G-to-A substitution attributable to cytidine deamination induced by APOBEC3G, APOBEC3F, or both enzymes were present in individuals carrying proviruses with completely or partly defective Vif variants. Natural variation in Vif function may result in selective and partial neutralization of cytidine deaminases and thereby promote viral sequence diversification within HIV-1 infected individuals

HIV therapy by a combination of broadly neutralizing antibodies in humanized mice

Human antibodies to human immunodeficiency virus-1 (HIV-1) can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice. Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy the longer half-life of antibodies led to control of viraemia for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in humanized mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals