Evolutionary conservation of an ancient retroviral gagpol gene in Artiodactyla.

The genomes of mammals contain fingerprints of past infections by ancient retroviruses that invaded the germline of their ancestors. Most of these endogenous retroviruses (ERVs) contain only remnants of the original retrovirus; however, on rare occasions, ERV genes can be co-opted for a beneficial host function. While most studies of co-opted ERVs have focused on envelope genes, including the syncytins that function in placentation, there are examples of co-opted gag genes including one we recently discovered in simian primates. Here, we searched for other intact gag genes in non-primate mammalian lineages. We began by examining the genomes of extant camel species, which represent a basal lineage in the order Artiodactyla. This identified a gagpol gene with a large open reading frame (ORF) (>3,500 bp) in the same orthologous location in Artiodactyla species but that is absent in other mammals. Thus, this ERV was fixed in the common ancestor of all Artiodactyla at least 64 million years ago. The amino acid sequence of this gene, termed ARTgagpol, contains recognizable matrix, capsid, nucleocapsid, and reverse transcriptase domains in ruminants, with an RNase H domain in camels and pigs. Phylogenetic analysis and structural prediction of its reverse transcriptase and RNase H domains groups ARTgagpol with gammaretroviruses. Transcriptomic analysis shows ARTgagpol expression in multiple tissues suggestive of a co-opted host function. These findings identify the oldest and largest ERV-derived gagpol gene with an intact ORF in mammals, an intriguing milestone in the co-evolution of mammals and retroviruses. IMPORTANCE Retroviruses are unique among viruses that infect animals as they integrate their reverse-transcribed double-stranded DNA into host chromosomes. When this happens in a germline cell, such as sperm, egg, or their precursors, the integrated retroviral copies can be passed on to the next generation as endogenous retroviruses (ERVs). On rare occasions, the genes of these ERVs can be domesticated by the host. In this study we used computational similarity searches to identify an ancient ERV with an intact viral gagpol gene in the genomes of camels that is also found in the same genomic location in other even-toed ungulates suggesting that it is at least 64 million years old. Broad tissue expression and predicted preservation of the reverse transcriptase fold of this protein suggest that it may be domesticated for a host function. This is the oldest known intact gagpol gene of an ancient retrovirus in mammals.

Cross-species transmission of an ancient endogenous retrovirus and convergent co-option of its envelope gene in two mammalian orders.

Endogenous retroviruses (ERVs) found in vertebrate genomes are remnants of retroviral invasions of their ancestral species. ERVs thus represent molecular fossil records of ancient retroviruses and provide a unique opportunity to study viral-host interactions, including cross-species transmissions, in deep time. While most ERVs contain the mutated remains of the original retrovirus, on rare occasions evolutionary selection pressures lead to the co-option/exaptation of ERV genes for a host function. Here, we report the identification of two ancient related non-orthologous ERV env genes, ARTenvV and CARenvV, that are preserved with large open reading frames (ORFs) in the mammalian orders Artiodactyla and Carnivora, respectively, but are not found in other mammals. These Env proteins lack a transmembrane motif, but phylogenetic analyses show strong sequence preservation and positive selection of the env surface ORF in their respective orders, and transcriptomic analyses show a broad tissue expression pattern for both ARTenvV and CARenvV, suggesting that these genes may be exapted for a host function. Multiple lines of evidence indicate that ARTenvV and CARenvV were derived from an ancient ancestral exogenous gamma-like retrovirus that was independently endogenized in two mammalian orders more than 60 million years ago, which roughly coincides with the K-Pg mass extinction event and subsequent mammalian diversification. Thus, these findings identify the oldest known retroviral cross-ordinal transmission of a gamma-like retrovirus with no known extant infectious counterpart in mammals, and the first discovery of the convergent co-option of an ERV gene derived from the same ancestral retrovirus in two different mammalian orders.

Patterns of Coevolutionary Adaptations across Time and Space in Mouse Gammaretroviruses and Three Restrictive Host Factors.

The classical laboratory mouse strains are genetic mosaics of three Mus musculus subspecies that occupy distinct regions of Eurasia. These strains and subspecies carry infectious and endogenous mouse leukemia viruses (MLVs) that can be pathogenic and mutagenic. MLVs evolved in concert with restrictive host factors with some under positive selection, including the XPR1 receptor for xenotropic/polytropic MLVs (X/P-MLVs) and the post-entry restriction factor Fv1. Since positive selection marks host-pathogen genetic conflicts, we examined MLVs for counter-adaptations at sites that interact with XPR1, Fv1, and the CAT1 receptor for ecotropic MLVs (E-MLVs). Results describe different co-adaptive evolutionary paths within the ranges occupied by these virus-infected subspecies. The interface of CAT1, and the otherwise variable E-MLV envelopes, is highly conserved; antiviral protection is afforded by the Fv4 restriction factor. XPR1 and X/P-MLVs variants show coordinate geographic distributions, with receptor critical sites in envelope, under positive selection but with little variation in envelope and XPR1 in mice carrying P-ERVs. The major Fv1 target in the viral capsid is under positive selection, and the distribution of Fv1 alleles is subspecies-correlated. These data document adaptive, spatial and temporal, co-evolutionary trajectories at the critical interfaces of MLVs and the host factors that restrict their replication.

The Oldest Co-opted gag Gene of a Human Endogenous Retrovirus Shows Placenta-Specific Expression and Is Upregulated in Diffuse Large B-Cell Lymphomas.

Vertebrate genomes contain endogenous retroviruses (ERVs) that represent remnants of past germline infections by ancient retroviruses. Despite comprising 8% of the human genome, the human ERVs (HERVs) do not encode a replication competent retrovirus. However, some HERV genes have been co-opted to serve host functions, most notably the viral envelope-derived syncytins involved in placentation. Here, we identify the oldest HERV intact gag gene with an open reading frame, gagV1. Its provirus contains an intact env, envV1, and the first open reading frame found in an HERV gag leader, pre-gagV1, which encodes a novel protein. This HERV is linked to a related gag gene, gagV3, and these three genes all show patterns of evolutionary conservation in primates. gagV1 and pre-gagV1 orthologs are present in all simian primate lineages indicating that this HERV entered the germline of the common simian primate ancestor at least 43 Ma, whereas gagV3 is found in Old and New World monkeys. gagV1 and gagV3 have undergone recurrent gene conversion events and positive selection. Expression of gagV1, gagV3, and pre-gagV1 is restricted to the placenta in humans and macaques suggesting co-option for placenta-specific host functions. Transcriptomic analysis of human tumors also found upregulated levels of gagV1 transcripts in diffuse large B-cell lymphomas. These findings suggest that these HERV-V genes may be useful markers for the most common type of non-Hodgkin's lymphoma and that they may have contributed to the successive domestications of env and gag genes in eutherians involved in the ongoing ERV-driven evolution of the placenta.

Retroviral Restriction Factors and Their Viral Targets: Restriction Strategies and Evolutionary Adaptations.

The evolutionary conflict between retroviruses and their vertebrate hosts over millions of years has led to the emergence of cellular innate immune proteins termed restriction factors as well as their viral antagonists. Evidence accumulated in the last two decades has substantially increased our understanding of the elaborate mechanisms utilized by these restriction factors to inhibit retroviral replication, mechanisms that either directly block viral proteins or interfere with the cellular pathways hijacked by the viruses. Analyses of these complex interactions describe patterns of accelerated evolution for these restriction factors as well as the acquisition and evolution of their virus-encoded antagonists. Evidence is also mounting that many restriction factors identified for their inhibition of specific retroviruses have broader antiviral activity against additional retroviruses as well as against other viruses, and that exposure to these multiple virus challenges has shaped their adaptive evolution. In this review, we provide an overview of the restriction factors that interfere with different steps of the retroviral life cycle, describing their mechanisms of action, adaptive evolution, viral targets and the viral antagonists that evolved to counter these factors.

Evolution of the rodent Trim5 cluster is marked by divergent paralogous expansions and independent acquisitions of TrimCyp fusions.

Evolution of cellular innate immune genes in response to viral threats represents a rich area of study for understanding complex events that shape mammalian genomes. One of these genes, TRIM5, is a retroviral restriction factor that mediates a post-entry block to infection. Previous studies on the genomic cluster that contains TRIM5 identified different patterns of gene amplification and the independent birth of CypA gene fusions in various primate species. However, the evolution of Trim5 in the largest order of mammals, Rodentia, remains poorly characterized. Here, we present an expansive phylogenetic and genomic analysis of the Trim5 cluster in rodents. Our findings reveal substantial evolutionary changes including gene amplifications, rearrangements, loss and fusion. We describe the first independent evolution of TrimCyp fusion genes in rodents. We show that the TrimCyp gene found in some Peromyscus species was acquired about 2 million years ago. When ectopically expressed, the P. maniculatus TRIMCyp shows anti-retroviral activity that is reversed by cyclosporine, but it does not activate Nf-κB or AP-1 promoters, unlike the primate TRIMCyps. These results describe a complex pattern of differential gene amplification in the Trim5 cluster of rodents and identify the first functional TrimCyp fusion gene outside of primates and tree shrews.

Ancient Evolutionary Origin and Positive Selection of the Retroviral Restriction Factor Fv1 in Muroid Rodents.

The laboratory mouse Fv1 gene encodes a retroviral restriction factor that mediates resistance to murine leukemia viruses (MLVs). Sequence similarity between Fv1 and the gag protein of the murine endogenous retrovirus L (MuERV-L) family of ERVs suggests that Fv1 was coopted from an ancient provirus. Previous evolutionary studies found Fv1 orthologs only in the genus Mus Here, we describe identification of orthologous Fv1 sequences in several species belonging to multiple families of rodents outside the genus Mus We show that these Fv1 orthologs are in the same region of conserved synteny, between the genes Miip and Mfn2, suggesting a minimum insertion time of 45 million years for the ancient progenitor of Fv1 Our analysis also revealed that Fv1 was not detectable or heavily mutated in some lineages in the superfamily Muroidea, while, in concert with previous findings in the genus Mus, we found strong evidence of positive selection of Fv1 in the African clade in the subfamily Muridae Residues identified as evolving under positive selection include those that have been previously found to be important for restriction of multiple retroviral lineages. Taken together, these findings suggest that the evolutionary origin of Fv1 substantially predates Mus evolution, that the rodent Fv1 has been shaped by lineage-specific differential selection pressures, and that Fv1 has long been evolving under positive selection in the rodent family Muridae, supporting a defensive role that significantly antedates exposure to MLVs.IMPORTANCE Retroviruses have adapted to living in concert with their hosts throughout vertebrate evolution. Over the years, the study of these relationships revealed the presence of host proteins called restriction factors that inhibit retroviral replication in host cells. The first of these restriction factors to be identified, encoded by the Fv1 gene found in mice, was thought to have originated in the genus Mus In this study, we utilized genome database searches and DNA sequencing to identify Fv1 copies in multiple rodent lineages. Our findings suggest a minimum time of insertion into the genome of rodents of 45 million years for the ancestral progenitor of Fv1 While Fv1 is not detectable in some lineages, we also identified full-length orthologs showing signatures of a molecular "arms race" in a family of rodent species indigenous to Africa. This finding suggests that Fv1 in these species has been coevolving with unidentified retroviruses for millions of years.

Large-Scale Arrayed Analysis of Protein Degradation Reveals Cellular Targets for HIV-1 Vpu.

Accessory proteins of lentiviruses, such as HIV-1, target cellular restriction factors to enhance viral replication. Systematic analyses of proteins that are targeted for degradation by HIV-1 accessory proteins may provide a better understanding of viral immune evasion strategies. Here, we describe a high-throughput platform developed to study cellular protein stability in a highly parallelized matrix format. We used this approach to identify cellular targets of the HIV-1 accessory protein Vpu through arrayed coexpression with 433 interferon-stimulated genes, followed by differential fluorescent labeling and automated image analysis. Among the previously unreported Vpu targets identified by this approach, we find that the E2 ligase mediating ISG15 conjugation, UBE2L6, and the transmembrane protein PLP2 are targeted by Vpu during HIV-1 infection to facilitate late-stage replication. This study provides a framework for the systematic and high-throughput evaluation of protein stability and establishes a more comprehensive portrait of cellular Vpu targets.

Characterization of resistance to rhabdovirus and retrovirus infection in a human myeloid cell line.

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.

The nature of the N-terminal amino acid residue of HIV-1 RNase H is critical for the stability of reverse transcriptase in viral particles.

UNLABELLED: Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is synthesized and packaged into the virion as a part of the GagPol polyprotein. Mature RT is released by the action of viral protease. However, unlike other viral proteins, RT is subject to an internal cleavage event leading to the formation of two subunits in the virion: a p66 subunit and a p51 subunit that lacks the RNase H domain. We have previously identified RNase H to be an HIV-1 protein that has the potential to be a substrate for the N-end rule pathway, which is an ubiquitin-dependent proteolytic system in which the identity of the N-terminal amino acid determines the half-life of a protein. Here we examined the importance of the N-terminal amino acid residue of RNase H in the early life cycle of HIV-1. We show that changing this residue to an amino acid structurally different from the conserved residue leads to the degradation of RT and, in some cases, integrase in the virus particle and this abolishes infectivity. Using intravirion complementation and in vitro protease cleavage assays, we show that degradation of RT in RNase H N-terminal mutants occurs in the absence of active viral protease in the virion. Our results also indicate the importance of the RNase H N-terminal residue in the dimerization of RT subunits. IMPORTANCE: HIV-1 proteins are initially made as part of a polyprotein that is cleaved by the viral protease into the proteins that form the virus particle. We were interested in one particular protein, RNase H, that is cleaved from reverse transcriptase. In particular, we found that the first amino acid of RNase H never varied in over 1,850 isolates of HIV-1 that we compared. When we changed the first amino acid, we found that the reverse transcriptase in the virus was degraded. While other studies have implied that the viral protease can degrade mutant RT proteins, we show here that this may not be the case for our mutants. Our results suggest that the presence of active viral protease is not required for the degradation of RT in RNase H N-terminal mutants, suggesting a role for a cellular protease in this process.

The N-end rule and retroviral infection: no effect on integrase.

BACKGROUND: Integration of double stranded viral DNA is a key step in the retroviral life cycle. Virally encoded enzyme, integrase, plays a central role in this reaction. Mature forms of integrase of several retroviruses (i.e. HIV-1 and MLV) bear conserved destabilizing N-terminal residues of the N-end rule pathway - a ubiquitin dependent proteolytic system in which the N-terminal residue of a protein determines its half life. Substrates of the N-end rule pathway are recognized by E3 ubiquitin ligases called N-recognins. We have previously shown that the inactivation of three of these N-recognins, namely UBR1, UBR2 and UBR4 in mouse embryonic fibroblasts (MEFs) leads to increased stability of ectopically expressed HIV-1 integrase. These findings have prompted us to investigate the involvement of the N-end rule pathway in the HIV-1 life cycle. RESULTS: The infectivity of HIV-1 but not MLV was decreased in N-recognin deficient cells in which three N-recognins (UBR1, UBR2 and UBR4) were depleted. HIV-1 integrase mutants of N-terminal amino acids (coding for stabilizing or destabilizing residues) were severely impaired in their infectivity in both human and mouse cells. Quantitative PCR analysis revealed that this inhibition was mainly caused by a defect in reverse transcription. The decreased infectivity was independent of the N-end rule since cells deficient in N-recognins were equally refractory to infection by the integrase mutants. MLV integrase mutants showed no difference in their infectivity or intravirion processing of integrase. CONCLUSIONS: The N-end rule pathway impacts the early phase of the HIV-1 life cycle; however this effect is not the result of the direct action of the N-end rule pathway on the viral integrase. The N-terminal amino acid residue of integrase is highly conserved and cannot be altered without causing a substantial decrease in viral infectivity.

Dexamethasone induces dysferlin in myoblasts and enhances their myogenic differentiation.

Glucocorticoids are beneficial in many muscular dystrophies but they are ineffective in treating dysferlinopathy, a rare muscular dystrophy caused by loss of dysferlin. We sought to understand the molecular basis for this disparity by studying the effects of a glucocorticoid on differentiation of the myoblast cell line, C2C12, and dysferlin-deficient C2C12s. We found that pharmacologic doses of dexamethasone enhanced the myogenic fusion efficiency of C2C12s and increased the induction of dysferlin, along with specific myogenic transcription factors, sarcolemmal and structural proteins. In contrast, the dysferlin-deficient C2C12 cell line demonstrated a reduction in long myotubes and early induction of particular muscle differentiation proteins, most notably, myosin heavy chain. Dexamethasone partially reversed the defect in myogenic fusion in the dysferlin-deficient C2C12 cells. We hypothesize that a key therapeutic benefit of glucocorticoids may be the up-regulation of dysferlin as an important component of glucocorticoid-enhanced myogenic differentiation.