The phylogeny of orthoretroviral long terminal repeats (LTRs)

LTRs are sequence elements in retroviruses and retrotransposons which are difficult to align due to their variability. One way of handling such cases is to use Hidden Markov Models (HMMs). In this work HMMs of LTRs were constructed for three groups of orthoretroviruses: the betaretroviruslike human MMTV-like (HML) endogenous retroviruses, the lentiviruses, including HIV, and gammaretroviruslike human endogenous retroviruses (HERVs). The HMM-generated LTR alignments and the phylogenetic trees constructed from them were compared with trees based on alignments of the pol gene at the nucleic acid level. The majority of branches in the LTR and pol based trees had the same order for the three retroviral genera, showing that HMM methods are successful in aligning and constructing phylogenies of LTRs. The HML LTR tree deviated somewhat from the pol tree for the groups HML3, HML7 and HML6. Among the gammaretroviruslike proviruses, the exogenous Mouse Leukemia Virus (MLV) was highly related to HERV-T in the pol based tree, but not in the LTR based tree. Aside from these differences, the similarity between the trees indicates that LTRs and pol coevolved in a largely monophyletic way.     

Fighting Fire with Fire: Endogenous Retrovirus Envelopes as Restriction Factors

A considerable portion of vertebrate genomes are made up of endogenous retroviruses (ERVs). While aberrant or uncontrolled ERV expression has been perceived as a potential cause of disease, there is mounting evidence that some ERVs have become integral components of normal host development and physiology. Here, we revisit the longstanding concept that some of the gene products encoded by ERVs and other endogenous viral elements may offer to the host protection against viral infection. Notably, proteins produced from envelope (env) genes have been shown to act as restriction factors against related exogenous retroviruses in chickens, sheep, mice, and cats. Based on the proposed mode of restriction and the domain architecture of known antiretroviral env, we argue that many more env gene-derived restriction factors await discovery in vertebrate genomes, including the human genome.     

Interactions between exogenous and endogenous retroviruses

Retroviruses are distinguished from other viruses by several features. Notably, some retroviruses are present as normal elements in the genomes of virtually all vertebrates (endogenous proviruses). Others are exogenous, i.e. horizontally transmitted agents, many of which cause fatal diseases. The endogenous retroviruses are genetically transmitted and to a large extent their significance is uncertain. However, there is evidence suggesting that they contribute to the development of diseases in several animal species. Most importantly, some endogenous retroviruses are capable of interacting with exogenous counterparts through a variety of different mechanisms with serious consequences to the host. Conversely, others are advantageous in that they protect against exogenous retroviruses. In this review various types of interactions between endogenous and exogenous retroviruses are discussed, including receptor interference, recombination, phenotypic mixing, immunological interactions and heterologoustrans-activation.     

Parallel Germline Infiltration of a Lentivirus in Two Malagasy Lemurs

Retroviruses normally infect the somatic cells of their host and are transmitted horizontally, i.e., in an exogenous way. Occasionally, however, some retroviruses can also infect and integrate into the genome of germ cells, which may allow for their vertical inheritance and fixation in a given species; a process known as endogenization. Lentiviruses, a group of mammalian retroviruses that includes HIV, are known to infect primates, ruminants, horses, and cats. Unlike many other retroviruses, these viruses have not been demonstrably successful at germline infiltration. Here, we report on the discovery of endogenous lentiviral insertions in seven species of Malagasy lemurs from two different genera—Cheirogaleus and Microcebus. Combining molecular clock analyses and cross-species screening of orthologous insertions, we show that the presence of this endogenous lentivirus in six species of Microcebus is the result of one endogenization event that occurred about 4.2 million years ago. In addition, we demonstrate that this lentivirus independently infiltrated the germline of Cheirogaleus and that the two endogenization events occurred quasi-simultaneously. Using multiple proviral copies, we derive and characterize an apparently full length and intact consensus for this lentivirus. These results provide evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species and that primates have been exposed to lentiviruses for a much longer time than what can be inferred based on sequence comparison of circulating lentiviruses. The study sets the stage for an unprecedented opportunity to reconstruct an ancestral primate lentivirus and thereby advance our knowledge of host–virus interactions.     

A large number of the human microRNAs target lentiviruses, retroviruses, and endogenous retroviruses

Retroelements (including transposons, retrotransposons, retroviruses, and lentiviruses) make up a significant portion of eukaryotic genomes. Given their ability to mutate genes these mobile elements always present a threat to the integrity of the host genomes. Recent studies have revealed complex molecular mechanisms that silence the mutagenic ability of these RE as well strategically express the pieces of the incorporated RE that are utilized to silence human endogenous retroviruses (HERVs) or invading exogenous retroviruses (IERV). We have hypothesized that small endogenous RNA originally evolved to quell “foreign” IERV-genes and subsequently emerged into elaborate silencing systems that include RNA interference, miRNA-based gene regulation and other gene silencing mechanisms. Here, we present evidence that the replication of complex RE are most likely silenced or regulated by homologous miRNA that are found as a part of the cellular repertoire. We analyzed Homo sapiens miRNAs for possible target genetic sequences in selected HERVs and IERV found in humans and other large primates. We identified several miRNAs that have >80% sequence homology with human HERVs; -L, -W, and -K, and IERV like SIVcpz, HTLV-1, and HTLV-2. We found an inverse correlation between the numbers and relative degree of homology of miRNAs to the relative replication capacity of a specific RE. Therefore, larger numbers of miRNAs with greater degree of homology are found against the least active RE and the least numbers of miRNAs with smaller degree of homology are found against the most active RE (i.e. HERV-K). Implications of these observations in RE disease and therapy are discussed.     

Identification of a Full-Length cDNA for an Endogenous Retrovirus of Miniature Swine

Endogenous retroviruses of swine are a concern in the use of pig-derived tissues for xenotransplantation into humans. The nucleotide sequence of porcine endogenous retrovirus taken from lymphocytes of miniature swine (PERV-MSL) has been characterized. PERV-MSL is a type C retrovirus of 8,132 bp with the greatest nucleic acid sequence identity to gibbon ape leukemia virus and murine leukemia virus. Constitutive production of PERV-MSL RNA has been detected in normal leukocytes and in multiple organs of swine. The copy numbers of full-length PERV sequences per genome (approximately 8 to 15) vary among swine strains. The open reading frames for gag, pol, and env in PERV-MSL have over 99% amino acid sequence identity to those of Tsukuba-1 retrovirus and are highly homologous to those of endogenous retrovirus of cell line PK15 (PK15-ERV). Most of the differences in the predicted amino acid sequences of PK15-ERV and PERV-MSL are in the SU (cell attach- ment) region of env. The existence of these PERV clones will enable studies of infection by endogenous retroviruses in xenotransplantation.     

The Extraordinary Evolutionary History of the Reticuloendotheliosis Viruses

The reticuloendotheliosis viruses (REVs) comprise several closely related amphotropic retroviruses isolated from birds. These viruses exhibit several highly unusual characteristics that have not so far been adequately explained, including their extremely close relationship to mammalian retroviruses, and their presence as endogenous sequences within the genomes of certain large DNA viruses. We present evidence for an iatrogenic origin of REVs that accounts for these phenomena. Firstly, we identify endogenous retroviral fossils in mammalian genomes that share a unique recombinant structure with REVs—unequivocally demonstrating that REVs derive directly from mammalian retroviruses. Secondly, through sequencing of archived REV isolates, we confirm that contaminated Plasmodium lophurae stocks have been the source of multiple REV outbreaks in experimentally infected birds. Finally, we show that both phylogenetic and historical evidence support a scenario wherein REVs originated as mammalian retroviruses that were accidentally introduced into avian hosts in the late 1930s, during experimental studies of P. lophurae, and subsequently integrated into the fowlpox virus (FWPV) and gallid herpesvirus type 2 (GHV-2) genomes, generating recombinant DNA viruses that now circulate in wild birds and poultry. Our findings provide a novel perspective on the origin and evolution of REV, and indicate that horizontal gene transfer between virus families can expand the impact of iatrogenic transmission events.     

Mobile element <Emphasis Type="Italic">Gypsy</Emphasis> (Mdg4)—Endoparasyte of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: Regulation of transposition and infection characteristics

By the example of MDG4 (gypsy) of Drosophila melanogaster, characteristics of endogenous invertebrate retroviruses of the errantiviruses class (Errantiviridae, Metaviridae) are analyzed. Ways of the evolution of retroviruses of invertebrates together with possible mechanisms of their origin from retrotransposons via the addition of viral genes from other systematic groups are discussed. It has been demonstrated that the locus flamenco plays an important role in providing the internal immunity against MDG4 (gypsy) in Drosophila melanogaster.     

Spontaneous heteromerization of gammaretrovirus envelope proteins: a possible novel mechanism of retrovirus restriction

The env gene of gammaretroviruses encodes a glycoprotein conserved among diverse retroviruses, except for the domains involved in receptor binding. Here we show that pairs of gammaretrovirus envelope proteins (from Friend virus and GALV or xenotropic viruses) assemble into heteromers when coexpressed. This assembly results in a strong inhibition of infectivity. An unrelated envelope protein does not assemble in heteromers with the gammaretrovirus glycoproteins tested and does not affect their infectivity, demonstrating the specificity of the mechanism we describe. We propose that the numerous copies of endogenous retroviral env genes conserved within mammalian genomes act as restriction factors against infectious retroviruses.     

Insertional polymorphisms of full-length endogenous retroviruses in humans

Human endogenous retrovirus K (HERV-K) is distinctive among the retroviruses in the human genome in that many HERV-K proviruses were inserted into the human germline after the human and chimpanzee lineages evolutionarily diverged [1, 2]. However, all full-length endogenous retroviruses described to date in humans are sufficiently old that all humans examined were homozygous for their presence [1]. Moreover, none are intact; all have lethal mutations [1, 3, 4]. Here, we describe the first endogenous retroviruses in humans for which both the full-length provirus and the preintegration site alleles are shown to be present in the human population today. One provirus, called HERV-K113, was present in about 30% of tested individuals, while a second, called HERV-K115, was found in about 15%. HERV-K113 has full-length open reading frames (ORFs) for all viral proteins and lacks any nonsynonymous substitutions in amino acid motifs that are well conserved among retroviruses. This is the first such endogenous retrovirus identified in humans. These findings indicate that HERV-K remained capable of reinfecting humans through very recent evolutionary times and that HERV-K113 is an excellent candidate for an endogenous retrovirus that is capable of reinfecting humans today.     

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.     

Sequence Analysis of Mus dunni Endogenous Virus Reveals a Hybrid VL30/Gibbon Ape Leukemia Virus-Like Structure and a Distinct Envelope

Mus dunni endogenous virus (MDEV) can be activated from M. dunni cells by exposing the cells to hydrocortisone or 5-iodo-2′-deoxyuridine. Interference analysis has revealed that MDEV uses a receptor for cell entry that is different from those used by other murine retroviruses. The entire genome has now been sequenced, revealing a long terminal repeat (LTR)-gag-pol-env-LTR structure typical of simple retroviruses of the murine leukemia virus genus, with no additional open reading frames between env and the 3′ LTR. The LTRs and other noncoding regions of MDEV are most closely related to those of VL30 elements, while the majority of the coding sequences are most closely related to those of gibbon ape leukemia virus. MDEV represents the first example of a naturally occurring, replication-competent virus with sequences closely related to VL30 elements. The U3 region of MDEV contains six nearly perfect 80-bp repeats and the beginning of a seventh, and the region expected to contain the packaging sequence contains approximately four imperfect 33-bp repeats. The receptor specificity domains of the envelope are unique among retroviruses and show no apparent similarity to regions of known proteins.     

Endogenous Viral Sequences from the Cape Golden Mole (Chrysochloris asiatica) Reveal the Presence of Foamy Viruses in All Major Placental Mammal Clades

Endogenous retroviruses provide important insights into the deep history of this viral lineage. Endogenous foamy viruses are thought to be very rare and only a few cases have been identified to date. Here we report a novel endogenous foamy virus (CaEFV) within the genome of the Cape golden mole (Chrysochloris asiatica). The identification of CaEFV reveals the presence of foamy virus in the placental mammal superorder Afrotheria. Phylogenetic analyses place CaEFV basal to other foamy viruses of Eutherian origin, suggesting an ancient codivergence between foamy virus and placental mammals. These findings have implications for understanding the long-term evolution, diversity, and biology of retroviruses.     

A role for endogenous retroviral sequences in the regulation of lymphocyte activation

The genomes of most vertebrates contain numerous retroviral sequences, the great majority of which are non-infectious. These endogenous retroviral sequences are transcribed and translated in many host tissues, and are induced by mitogens. The function, if any, of endogenous retroviruses has been unclear. The transmembrane envelope proteins of some infectious type C retroviruses suppress lymphocyte activation, but it is unknown whether any endogenous type C retroviruses share this suppressive activity. To study the possible effects of murine endogenous retroviral expression, specific antisense oligonucleotides were synthesized complementary to type C retroviral sequences, and were cultured with murine spleen cells. If any of these endogenous retroviruses are suppressing lymphocyte activation, then inhibiting such endogenous retroviral-mediated suppression with antisense might result in lymphocyte stimulation. Three classes of endogenous type C retroviral sequences may be distinguished by antisense oligonucleotides (based on their homology to infectious retroviruses): ecotropic, xenotropic, and mink cell focus-forming (MCF). Antisense oligonucleotides to endogenous MCF envelope gene (env) initiation regions caused: i) doubling or tripling of spleen cell RNA synthesis, and ii) marked increases in lymphocyte surface Ia and Ig expression relative to control oligonucleotides. Antisense oligos to xenotropic or ecotropic env sequences or to endogenous MCF non-envelope sequences had no effect. These data suggest that endogenous MCF sequences exert an inhibitory influence on the murine immune system. Because endogenous MCF expression is inducible by immune stimuli, such expression could constitute an inhibitory feedback circuit that participates in the regulation of immune homeostasis.     

Negative Selection by an Endogenous Retrovirus Promotes a Higher-Avidity CD4+ T Cell Response to Retroviral Infection

Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4⁺ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4⁺ T cells and diminished the response to FV infection. Surprisingly, however, CD4⁺ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4⁺ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4⁺ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4⁺ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity.     

Induced prion protein controls immune-activated retroviruses in the mouse spleen

The prion protein (PrP) is crucially involved in transmissible spongiform encephalopathies (TSE), but neither its exact role in disease nor its physiological function are known. Here we show for mice, using histological, immunochemical and PCR-based methods, that stimulation of innate resistance was followed by appearance of numerous endogenous retroviruses and ensuing PrP up-regulation in germinal centers of the spleen. Subsequently, the activated retroviruses disappeared in a PrP-dependent manner. Our results reveal the regular involvement of endogenous retroviruses in murine immune responses and provide evidence for an essential function of PrP in the control of the retroviral activity. The interaction between PrP and ubiquitous endogenous retroviruses may allow new interpretations of TSE pathophysiology and explain the evolutionary conservation of PrP.     

Genetic Analysis of the Rat Leukemia Virus: Influence of Viral Sequences in Transduction of the c-ras Proto-Oncogene and Expression of Its Transforming Activity

The rat leukemia virus (RaLV) is an endogenous retrovirus that is spontaneously released by Sprague-Dawley rat embryo cells. The overall structure of the RaLV genome resembles that of other simple, replication-competent retroviruses, but the sequence of the long terminal repeats (LTR) is unique and unrelated to the known retroviruses. Phylogenetically, the RaLV genome appears to be more closely related to the feline leukemia virus group of retroviruses than to the murine leukemia virus group. A remarkable feature of RaLV is that it is capable of transducing a ras proto-oncogene from rat tumor cells in the form of an acutely transforming virus, designated the Rasheed strain of the rat sarcoma virus (RaSV). With the exception of the c-ras sequence, the genomes of both RaLV and RaSV are collinear. The RaSV-encoded oncogene v-Ra-ras expresses a fusion protein with a molecular mass of 29 kDa, and it exhibits a unique structure that has not been described previously for any known virus. The 5′ end of this gene is derived from sequences encoding RaLV matrix followed by 20 bp derived from the U5 region of the RaLV LTR (RS-U5 element) which is joined at its 3′ end to sequences derived from all six (coding and noncoding) exons of the c-ras proto-oncogene at the 3′ end. This recombinational event represents a novel mechanism among the acutely transforming viruses that have been studied.     

Betaretroviral Envelope Subunits Are Noncovalently Associated and Restricted to the Mammalian Class

The structure of the transmembrane subunit (TM) of the retroviral envelope glycoprotein (Env) is highly conserved among most retrovirus genera and includes a pair of cysteines that forms an intramolecular disulfide loop within the ectodomain. Alpha-, gamma-, and deltaretroviruses have a third cysteine, adjacent to the loop, which forms a disulfide bond between TM and the surface subunit (SU) of Env, while lentiviruses, which have noncovalently associated subunits, lack this third cysteine. The Betaretrovirus genus includes Jaagsiekte sheep retrovirus (JSRV) and mouse mammary tumor virus (MMTV), as well as many endogenous retroviruses. Envelope subunit association had not been characterized in the betaretroviruses, but lack of a third cysteine in the TM ectodomain suggested noncovalently associated subunits. We tested the Env proteins of JSRV and MMTV, as well as human endogenous retrovirus K (HERV-K)108—a betaretrovirus-like human endogenous retrovirus—for intersubunit bonding and found that, as in the lentiviruses, the Env subunits lack an intersubunit disulfide bond. Since these results suggest that the number of cysteines in the TM loop region readily distinguishes between covalent and noncovalent structure, we surveyed endogenous retroviral TM sequences in the genomes of vertebrates represented in public databases and found that (i) retroviruses with noncovalently associated subunits have been present during all of anthropoid evolution and (ii) the noncovalent env motif is limited to mammals, while the covalent type is found among five vertebrate classes. We discuss implications of these findings for retroviral evolution, cross-species transmissions, and recombination events involving the env gene.