Retroviruses infect a broad range of vertebrate hosts that includes amphibians, reptiles, fish, birds and mammals. In addition, a typical vertebrate genome contains thousands of loci composed of ancient retroviral sequences known as endogenous retroviruses (ERVs). ERVs are molecular remnants of ancient retroviruses and proof that the ongoing relationship between retroviruses and their vertebrate hosts began hundreds of millions of years ago. The long-term impact of retroviruses on vertebrate evolution is twofold: first, as with other viruses, retroviruses act as agents of selection, driving the evolution of host genes that block viral infection or that mitigate pathogenesis, and second, through the phenomenon of endogenization, retroviruses contribute an abundance of genetic novelty to host genomes, including unique protein-coding genes and cis-acting regulatory elements. This Review describes ERV origins, their diversity and their relationships to retroviruses and discusses the potential for ERVs to reveal virus–host interactions on evolutionary timescales. It also describes some of the many examples of cellular functions, including protein-coding genes and regulatory elements, that have evolved from ERVs.
Retrovirus virions contain RNA copies of the viral genome. Upon entry into a target cell, these are reverse transcribed into a double-stranded DNA molecule and integrated into the genomic DNA of the host cell. The resulting provirus contains the promoters and regulatory elements required for transcription of viral RNA and encodes all the structural proteins and enzymes necessary for assembling progeny virions. Retroviruses typically infect somatic tissues; however, as a retrovirus spreads in a host population, there is an unknown but finite probability that integration may occur in germline cells or in the precursors of germline cells, resulting in production of host gametes carrying proviruses as novel insertions. Upon entering the host gene pool in this way, a provirus is known as an endogenous retrovirus (ERV) and is fated for either loss or fixation depending on the vagaries of random genetic drift and natural selection (Fig. 1). An ERV may also increase in copy number by various post-endogenization mechanisms. Thus, ERVs are genetic loci whose ultimate origins trace back to exogenously replicating retroviruses, regardless of whether they retain the capacity to express infectious virions. Indeed, the vast majority of ERVs are defective for viral gene expression as a consequence of mutations accumulated across thousands to millions of years of vertebrate evolution.
Endogenization is not an essential property of any known retrovirus, and germline insertion is probably very rare relative to infection of somatic tissues. Importantly, the ability to replicate and spread in germline cells is not a prerequisite for endogenization. Only the early stages of the retroviral life cycle (entry, reverse transcription and integration) are necessary for provirus biogenesis, and all viral components essential for completing these steps are provided by the incoming virion — neither de novo viral genome synthesis nor expression of viral genes is required to produce an integrated provirus. Nonetheless, over the span of millions of years, the genomes of vertebrates have accumulated thousands and, in some cases, hundreds of thousands of ERV loci. This vast molecular archive of ancient, extinct retroviruses has captured the attention of virologists and evolutionary biologists interested in the impact of viruses on the evolution of their vertebrate hosts1,2,3,4,5,6,7. In addition, because they are found in virtually all vertebrate genomes, ERVs may be expressed in many commonly used cell lines, tissues and model organisms, potentially compromising interpretation of experimental results, contaminating preparations of biological and pharmacological reagents and vaccines8,9, complicating the use of animal organs for xenotransplantation10 and, perhaps, contributing to human disease11,12. Moreover, ERV expression can be induced by a variety of conditions, including infection with viruses such as HIV or exposure to epigenetic modifying drugs13, and studies in cell culture and laboratory mice have documented the potential for recombination, either between ERVs or between ERVs and exogenous retroviruses, to produce viral strains with novel biological and pathogenic properties14,15,16,17. This Review describes ERVs and their relationship to exogenous retroviruses, highlights the ways in which ERVs aid our understanding of the origins and evolution of retroviruses, discusses advances in the reconstitution and functional characterization of ancient ERV genes and provides a virological perspective on the contributions of ERVs to cellular functions.
Diversity of endogenous retroviruses
All retroviruses have a similar genome structure (Fig. 2). Reverse transcription and integration result in a provirus of approximately 5–10 kb, comprising identical long-terminal repeats (LTRs) with the viral genes arrayed between them. LTRs contain the primary promoter and regulatory elements for provirus expression, as well as the cis-acting motifs required for integration. A common set of genes includes gag, which encodes the structural proteins that make up the virion core; pro, which encodes the viral protease; pol, which encodes the viral replicative enzymes reverse transcriptase (RT) and integrase (IN); and env, which encodes the glycoprotein complex that governs receptor-mediated fusion and entry. Retroviruses vary considerably in the number and genomic position of noncanonical accessory genes.
LTRs consist of three regions: from 5ʹ to 3ʹ, these are U3, R and U5 (Fig. 2b). R is repeated at both ends of the viral RNA, whereas U5 and U3 are present as one copy each. The process of reverse transcription duplicates U5 and U3 to produce identical LTRs at both ends of the DNA provirus. The U3–R junction corresponds to the transcription start site (TSS) in the 5ʹ LTR, whereas the R–U5 junction corresponds to the 3ʹ end of the proviral transcripts in the 3ʹ LTR. U3 contains various motifs that interact with the regulatory milieu of the host cell and governs provirus expression; its length varies between different retroviruses (~190–1,200 bases) and comprises a dense and highly variable cluster of enhancer and promoter elements18,19. The variations in U3 of different retroviruses reflect differences in cellular or tissue tropism and host range.
ERVs originate as integrated proviruses and can range from complete proviruses to highly fragmented remnants of proviruses. Even where substantial portions of gag, pro, pol and env remain, these are often inactive owing to the accumulation of substitutions, deletions and insertions. The degree of sequence degradation correlates approximately with the age of the provirus (that is, the amount of time that has passed since germline insertion). A majority of ERVs exist as solo-LTRs produced by homologous recombination between the 5ʹ and 3ʹ LTRs. Solo-LTR formation deletes all internal sequences, including the viral genes20. LTRs are the most variable sequences in the retroviral genome, and there is little or no resemblance between the LTRs of retroviruses from different genera18,19. Consequently, annotating solo-LTRs in genome assemblies often depends on an association with a known retrovirus or previously characterized ERV, although query-independent identification of LTRs has been reported18,19.
The presence of ERV sequences in genomic DNA was first confirmed more than 50 years ago21. In the years that followed, ERV loci were detected and characterized first by hybridization methods and later by cloning or PCR and were found in the genomes of a wide range of vertebrate species. Whole-genome sequencing and related computational tools accelerated the discovery and phylogenetic analysis of ERV loci, permitting detailed comparisons to extant retroviruses. ERV loci within a genome can be clustered into groups of related elements on the basis of sequence5,22,23. These groups may reflect multiple germline insertions by the same species of retrovirus but can also result from different post-endogenization amplification mechanisms24,25. These include activation and expression of an ERV locus resulting in particles that reinfect germline cells and insert new copies of the element; infection or retrotransposition in trans, whereby ERV transcripts are packaged, copied and integrated by another virus or transposable element; or expansions of chromosomal DNA segments that contain ERVs (for example, segmental duplications).
Reverse transcriptase amino acid sequences are highly conserved and readily aligned across the entire taxonomic range of known reverse-transcribing viruses and retrotransposons and are useful for reconstructing deep phylogenetic relationships. ERVs are easily incorporated into such analyses, either directly or after in silico reconstruction of RT-coding sequences. RT-based phylogenies of the family Retroviridae contain three major branches, and taxa comprising these branches are sometimes referred to as class I, II or III5,23,26. As more vertebrate genomes are assembled, incorporating larger numbers of ERVs has not drastically changed the overall topology — most retroviral and ERV RT sequences analysed to date cluster within the three main branches5,27. Retrovirus phylogenies can also be based on the conserved ectodomain of the transmembrane (TM) subunit of the viral envelope glycoprotein (Env), and discrepancies between RT and TM phylogenies can reveal lineages that originated by recombination between distantly related retroviruses28. The number of unique ERV lineages extracted from genome data now exceeds the number of distinct retroviruses that have been classified by the International Committee on Taxonomy of Viruses29. Incorporating these lineages into retroviral taxonomy will likely require creating additional genera within Retroviridae, some of which may consist mostly or exclusively of extinct retrovirus species30.
ERVs and retroviruses interleave in RT-based phylogenies, indicating that the phenomenon of endogenization is not unique to any particular type of retrovirus. However, there are notable differences in the degree to which different types of ERV are represented in vertebrate genomes. For example, ERVs related to gammaretroviruses are abundant in the genomes of a wide variety of vertebrate species31,32,33, whereas ERVs related to deltaretroviruses have only been identified in the genomes of Miniopterus and Rhinolophus bats34,35. ERVs related to lentiviruses are also rare and, thus far, have only been found in the genomes of a small number of mammalian species, none of which is known to host extant lentiviruses36,37,38,39,40,41,42. Differences in frequency and distribution of different types of ERV have not been explained but may reflect biological differences that influence the probability of endogenization. For example, a retrovirus that can infect germline cells as the result of broad tissue tropism or by virtue of being specifically adapted to germline cells would have a higher probability of producing heritable proviruses. Conversely, ERVs of viruses whose expression is intrinsically cytotoxic might be selected against and less likely to persist in the germ line.
Insights into ancient retroviruses
ERVs can be exploited to study the natural history of viruses and their hosts, revealing the extent to which vertebrate evolution has been impacted by retroviruses and providing insights relevant to the study of modern viruses. For example, a comparison of human endogenous retrovirus K HML-2 (HERV-K(HML-2)) loci found in human, Neanderthal and Denisovan genomes reflects the spread of an ancient betaretrovirus among the ancestors of modern humans43,44,45, while the discovery of unfixed, largely intact HERV-K(HML2)-related proviruses in gorillas raises the possibility that some populations may still harbour infectious virus46. Ancient ERVs in lemur and rabbit genomes are missing links that help clarify the relationship between divergent species of modern lentiviruses37. Similarly, 3D structures of ancient lentiviral capsid proteins have been resolved and compared with the corresponding structures of modern relatives, such as HIV-1 (ref.47). Ancient spumavirus-related ERVs suggest that retroviruses may have colonized marine animals of the Palaeozoic (more than 450 million years ago)48, and ERVs have been used to trace the emergence and spread of a gammaretrovirus during the Oligocene49. Reconstructed ERV sequences reveal extensive patterns of cross-species transmission of ancient viruses and broaden the known host ranges of modern viral groups32,49,50,51,52. ERV analysis has also revealed a striking difference in the rates at which viruses evolve over long versus short timescales, a major problem when applying molecular clock calculations to viral taxa2,53. Host populations with young (unfixed) ERVs, such as cervid endogenous gammaretrovirus (CrERVγ) elements in mule deer, help shed light on the earliest stages of the endogenization process54. This is particularly true for cases in which the related exogenous agent is still extant and potentially pathogenic, such as koala retrovirus (KoRV) in Australian koalas55.
A major challenge in such studies is accurate reconstruction of ancestral viral sequences from ERV data. Families of related ERV loci are convenient for generating and fine-tuning ancestral sequences by consensus49 or for inferring ancestral states by phylogenetic analysis56,57,58. ERVs are also uniquely amenable to molecular clock analysis59,60, which is useful for estimating integration times61,62 and for dating the emergence and spread of ancient retroviruses40,42,49,63,64 (Box 1).
Functional hypotheses can also be tested by biomolecular characterization of reconstituted ERV genes (Fig. 3). For example, promoters and regulatory elements can be studied using standard reporter assays65,66; retrotransposition can be detected and quantified using a sensitive cell culture assay58,67,68,69; and reconstituted proteins can be studied in the context of infection using pseudotyped particles or by replacing discrete domains in the polyproteins of replication-competent retroviruses with the homologous ERV domains70,71,72. Other viral platforms are also useful. For example, a rhabdovirus was engineered to express an ancient ERV Env in place of its own glycoprotein, creating a tool to delineate the viral entry pathway and to identify the cellular cofactors likely to have been used by the extinct virus73,74.
Reconstituted ERV proteins have been used to identify the entry receptors for two ancient retroviruses57,75 and to test the sensitivity of ancient viruses to host defence factors76. Reconstituted virus-like particles related to HERV-K(HML2) loci have been used to examine tropism, to test sensitivity to innate immune effectors and to reveal differences between genome-wide integration site preferences (in cell culture) and the distribution of HERV-K(HML2) loci in the human genome70,77,78,79,80.
There are now many examples of ERV loci that have evolved to provide important cellular functions, attracting the attention of researchers from various fields including virology, genome biology, population genetics and evolutionary developmental biology. In this regard, the past 100 years of research on retroviruses have provided a wealth of insight, as well as the various assays described above, that can be used to explore how retroviruses and ERVs have influenced the evolution of vertebrate genes and genomes.
Exaptation of endogenous retroviruses
Gould and Vrba coined the term exaptation to be used when referring to an adaptation that fulfils a new function distinct from its originally selected function81. They discussed, among other examples, repetitive DNA, including transposable elements, as a special class of sequences available for exaptation81. The idea that transposable elements may have roles in gene regulation was proposed in the mid-1950s by McClintock82 and was incorporated into an early hypothetical model of gene regulation83. ERVs are often categorized as transposable elements and are related to LTR retrotransposons (Box 2). However, the ultimate origins of ERVs are exogenous retroviruses, whose sequences reflected adaptation to a wide variety of vertebrate hosts and a spectrum of cellular niches. This distinctive natural history may contribute to the exaptive potential of ERVs, connecting the biology of rapidly evolving, exogenous retroviruses to the co-opted functions of their germline counterparts.
Exaptation of Env proteins
Most examples of exaptation of ERV-coding sequences involve env genes. The primary viral function of Env glycoproteins is to facilitate entry into host cells, which involves binding to cell surface receptors and driving fusion of the virion and cellular membranes (Fig. 4). For many retroviruses, expression of Env also interferes with cell surface expression of the receptor, rendering the cell resistant to reinfection — a phenomenon known as superinfection interference84,85.
Well-documented examples of env exaptation fall into two distinct categories: the first comprises syncytins, which are ERV-encoded Env proteins that function in mammalian placental morphogenesis7, and the second comprises ERV Envs that confer resistance to exogenous viral infection through mechanisms analogous to superinfection interference86.
The placental syncytins are the focus of several recent reviews7,87,88. Briefly, these ERV-encoded glycoproteins drive fusion of cytotrophoblasts to form the multinucleate syncytiotrophoblast layer7. The underlying mechanism involves cell–cell fusion and is analogous to viral entry (which depends on receptor binding to trigger fusion of virion and cellular membranes) (Fig. 4). The syncytins are a striking example of convergent evolution, having originated independently across multiple mammalian lineages, including marsupials89, as well as in at least one species of live-bearing reptile90.
Syncytin function has been confirmed in mice91. However, because most reported syncytins arose independently in different mammalian clades, they are not homologues, and it is therefore risky to extrapolate results of mouse experiments to nonrodent species. Identifying ERV-encoded syncytins in nonmodel organisms is instead based on rigorous but indirect criteria7. These include conservation within a clade of related taxa, placenta-specific expression and fusogenicity in cell culture (Fig. 3c). In the case of human syncytins, additional histological and tissue-culture-based evidence is also consistent with the proposed function (reviewed elsewhere88). Confirming other syncytins may require additional experiments in representative nonmodel organisms or genetic association studies correlating variant syncytin alleles with relevant phenotypes. Finally, it remains possible that some of the syncytins have additional, as yet unrecognized, functions.
Whether the receptors used by syncytins are the same as those used by the originating retroviruses is difficult to establish — most syncytins are tens of millions of years old, and the retroviruses that produced them are probably extinct. However, there is a precedent for reconstituting Env proteins from ERV sequences and using these to identify the receptors used by ancient retroviruses57,75 (Fig. 3); similar approaches may be useful for establishing whether a syncytin and related ERVs shared the same receptor.
Env-mediated entry restriction
Viral interactions with host macromolecules fall into two broad categories: those exploited by viruses to ensure optimal fitness and those that have evolved to block infection. Host cell factors in the latter category are often referred to as restriction factors. Examples of restriction factors that inhibit replication of retroviruses include the APOBEC3 family DNA editing enzymes, tetherin (also known as BST2), SAMHD1 and TRIM5α92,93. Viral genes acquired by endogenization also have the potential to become restriction factors86. Among these restriction factors, the most common are ERV-encoded proteins that block viral entry through receptor interference.
In 1981, it was reported that three endogenous loci of chickens (EV3, EV6 and EV9) confer entry-level blocks to infection by avian leukosis virus, most likely by receptor interference94 (Fig. 4). The authors correctly predicted that similar functions would be found in other species known to harbour ERVs. The prototypical example of ERV-mediated entry restriction is the murine Fv4 gene (also known as Akvr-1). Fv4 was first defined as a locus conferring resistance to experimental infection of laboratory mice by ecotropic murine leukaemia virus (MLV) and subsequently was correlated with expression of a novel MLV-related Env protein95. A similar resistance phenotype was observed in a population of feral mice in California, United States96. Cloning of Fv4 revealed that the same gene was responsible for the observed resistance in both cases, and sequencing revealed that Fv4 comprises a defective MLV provirus that retains an intact env ORF but lacks most of the 5ʹ half of the provirus including the 5ʹ LTR97. Fv4 expression is instead regulated by cellular sequences adjacent to the insertion98. Fv4 was likely selected by virtue of its ability to block infection by ecotropic strains of MLV. Two additional examples of genes encoding Env-mediated restriction in mice, Rcmf and Rcmf2, confer resistance to polytropic MLV strains; as with Fv4, both genes are incapable of expressing infectious, replication-competent virus99,100 (Fig. 5).
Env glycoproteins are normally anchored in the viral and cellular membranes by a membrane-spanning domain in the TM subunit (Fig. 2). However, ERV-mediated entry restriction can also involve secreted Env. In such cases, the secreted proteins have mutations resulting in a premature truncation, thereby eliminating the membrane-spanning domain. For example, feline REFREX proteins are truncated Env proteins derived from endogenous feline leukaemia virus (FeLV) that block entry of exogenous FeLV101. A truncated Env in the human genome, encoded by the suppressyn gene (also known as ERVH48-1), binds the receptor ASCT2 (also known as ATB0) used by syncytin 1 and several retroviruses; thus, suppressyn may have evolved to block entry of a virus that uses ASCT2, as a negative regulator of syncytin 1 (ref.102), or both.
Could ERV genes have evolved to restrict viruses that are now extinct? Proof of principle can be accomplished through reconstruction and functional analysis of ERV env genes (Fig. 3). This was recently done to demonstrate the antiviral function of HsaHTenv, which encodes a fusion-defective HERV-T Env in the human genome57. To test the hypothesis that HsaHTenv expression results in entry restriction, a functional HERV-T Env (representing the ancestral retrovirus) was first reconstructed and then used to identify the corresponding receptor. Expression of native HsaHTenv was found to block infection by virions bearing functionally reconstituted HERV-T Env through receptor interference57.
More than two dozen env ORFs have been identified in the human genome103,104; for most of these, there is, as yet, no direct evidence that they confer resistance to retroviruses in vivo. Intriguingly, HIV-1 infection of primary human CD4+ T cells induces expression of HERV-K(HML2) loci105. Some HERV-K(HML2) loci encode intact env ORFs, and transfection and expression of at least one of these in laboratory cell lines inhibit production of infectious HIV-1 (ref.106). Inhibition is not due to receptor interference, raising the possibility that one or more of these loci may exert antiviral effects through a novel mechanism; whether inhibition manifests in vivo remains to be determined.
Highly conserved ERV env genes of unknown function
The oldest intact ERV env genes reported are the percomorf gene of ray-finned fish107 and the primate HEMO gene108. The preservation of these as intact ORFs for more than 100 million years reflects long-term purifying selection and strongly suggests that both genes are likely to encode novel cellular functions. The age and conservation of percomorf argue against an antiviral function and instead suggest that percomorf may represent a new category of exapted function involving receptor-mediated membrane fusion. HEMO lacks a furin cleavage site and a hydrophobic fusion-peptide, indicating that it cannot be a fusogen, although it may retain its receptor-binding activity. Characterization of the human homologue reveals that HEMO is expressed as a full-length Env protein, which is cleaved by an unknown cellular protease to release a truncated extracellular form108. The secreted form is detectable in the blood of pregnant women and in placental blood and tissues, but its functions remain unknown.
Exaptation and features of Env
A majority of retrovirus Env proteins belong to one of two types, the gamma-type and the beta-type33 (Fig. 2c). Intriguingly, ERV loci with gamma-type env sequences are widely distributed among vertebrate genomes, whereas beta-type env sequences are largely found in mammalian genomes28,33. The reason for these markedly different distributions is unknown.
Intriguingly, almost all known examples of Env exaptation involve gamma-type Envs, including all the mammalian syncytins. The reasons for this bias are also unknown, but certain features may predispose gamma-type Env to exaptation. Gamma-type Envs have a modular arrangement, with discrete receptor-binding domains (RBDs) within the amino-terminal half of the Env surface (SU) subunit109. One speculative possibility is that modularity uncouples evolution of receptor specificity from functions located outside of the RBD (that is, by recombination), allowing these to evolve independently. Additionally, the carboxyl termini of gammaretrovirus Envs suppress fusogenicity110,111. These short R peptides are removed by the viral protease after virion assembly such that only Env complexes present on mature virions are fusion competent110,111. However, immature Env complexes within the virus-infected cell are still able to bind their cognate receptors and mediate superinfection interference. Thus, by preventing spontaneous cell–cell fusion, R domains may enhance the probability of fixation of gamma-type ERV env genes. Additional mutations might be selected to prevent activation in trans (for example, by other gammaretroviruses). Indeed, several reported entry-blocking ERVs are fusion defective57,112,113. Similarly, ERV–Fc env ORFs found in the genomes of multiple mammals, including humans49,114, have defects that prevent fusion and preclude a syncytin-like function (K. Halm, personal communication). Discovery of additional entry-blocking Envs may establish whether loss of fusogenicity is a common feature of such loci and whether the loss is the result of drift or selection. By contrast, syncytins require both receptor-binding and membrane fusion activities to function, whereas features that prevent fusion should be eliminated or modified by selection. Indeed, this is the case for human syncytin 1, which has lost R-peptide-mediated regulation and can direct viral protease-independent fusion115. Whether similar adaptations are found in other syncytins remains to be determined.
There are relatively few reports of beta-type ERV Envs with exapted functions116. Perhaps, beta-type Envs contribute novel functions, distinct from those associated with gamma-type Envs. Retroviruses with beta-type env genes often encode additional ORFs overlapping env117, which could influence the selection of endogenized forms.
Exaptation of other ERV proteins
There are a few reports of exaptation involving gag and pol118. The prototypical example is the Fv1 gene of mice119, which confers resistance to MLV120. Fv1 is an endogenous gag gene related to ERV-L elements119,121; expression of Fv1 blocks incoming viral capsid cores shortly after entry. Fv1 orthologues have been identified in a broad range of rodent species, and the estimated insertion time is 45–50 million years ago122,123. Indeed, some Fv1 homologues restrict retroviruses unrelated to MLV124, suggesting that Fv1 does not recognize conserved amino acid motifs but may instead detect structurally conserved spatial patterns in the hexameric lattice typical of retroviral capsid cores125.
The EnJS56A1 locus of domestic sheep (Ovis aries) also encodes a Gag protein, which can act as a transdominant inhibitor of a related exogenous virus known as Jaagsiekte sheep retrovirus (JSRV)126,127. Unlike Fv1, which blocks MLV replication shortly after entry, EnJS56A1 acts at a late stage in the JSRV replication cycle, interfering with proper trafficking and assembly of progeny virions126,127.
Gag-mediated antiviral functions have not been reported for human ERVs, although HERV-K(HML2) Gag has been shown to inhibit HIV-1 in cell culture128, raising the possibility that one or more HERV-K(HML2) loci may encode a protein that confers a late-stage block to the lentiviral replication cycle. It is not yet known whether this effect manifests in vivo, and HERV-K(HML2) loci have not been identified in reported genetic surveys of HIV-positive cohorts or in cellular screens for HIV-1-interacting factors. It was predicted that several human proteins are structurally related to the retrovirus Gag and Gag–Pro–Pol polyproteins (although many are likely derived from LTR retrotransposons)129; one of these, ARC, assembles into capsid-like structures that are strikingly similar to retroviral capsid cores130,131. Another, SASPase, is structurally and functionally analogous to retroviral proteases132.
Evidence for accessory genes is sometimes present in ERVs, particularly those related to exogenous retroviruses with complex genomes34,36,37,38,133. In some cases, these bear little resemblance to the accessory genes of their exogenous relatives, and any viral or exaptive functions remain speculative. A possible example of accessory gene exaptation involves the Mls (also known as Mtv) genes of mice, which originate from mouse mammary tumour virus (MMTV) sag genes134. These encode superantigens that activate T cells135. Expression of different endogenous sag loci (Mls genes) result in clonal deletion of different cognate T cell subsets; by eliminating target cells that support viral infection and dissemination, Mls expression may provide resistance to exogenous MMTV strains of the same Sag specificity136.
Betaretroviruses encode proteins required for optimal expression of unspliced viral RNA137,138. This raises the possibility that ERV-encoded versions of these proteins could also affect cellular transcripts139. Several HERV-K(HML2) loci in the human genome have the potential to encode such a protein, an RNA transport factor known as REC140,141. REC binds the 3ʹ end of unspliced viral RNA through a REC-responsive element (RcRE) encoded in HERV-K(HML2) LTRs140,141, of which there are close to 1,000 in the human genome64. Interestingly, the Rev protein of HIV-1 can also bind the HERV-K(HML2) RcRE140,141. Direct evidence that Rec or Rev influences transport of cellular transcripts in vivo has not been reported but may be worthy of investigation.
Genomic signatures of exaptation
Initially, exapted ERV ORFs were identified by traditional means, for example, in seeking to explain a specific phenotype or by functional assays of candidate genes. Exapted ERV genes can be identified without a priori knowledge of a phenotype. For example, most syncytins and resistance-conferring env genes are in proviruses with disrupted gag, pro and pol genes. This reflects the degree to which the locus has accumulated random substitutions. The juxtaposition of an intact env ORF is therefore consistent with purifying selection focused on env (Fig. 5). Statistical tests of selection can also be applied, such as the dN:dS ratio (ω)142. The accumulation of silent changes (dS) sets a baseline expectation for drift, against which the accumulation of nonsynonymous changes can be evaluated. Ratios <1, =1 or >1 indicate purifying selection, drift and positive selection, respectively. Importantly, purifying selection and positive selection are not mutually exclusive; even for genes that have experienced positive selection, a majority of codons still evolve under purifying selection to maintain overall structure and function. Average ω values for percomorf and HEMO are <1, consistent with long-term purifying selection and strong indications that these genes encode functional proteins107,108. In contrast to percomorf and HEMO, analysis of Fv1 reveals a combination of long-term positive selection with periodic bouts of lineage-specific selection focused on residues involved in target specificity122,123, a combination typical of many antiretroviral proteins92,93. If there are insufficient taxa to calculate ω, one can also simulate neutral evolution of the ORF to derive a probability distribution for inactivating mutations57,143.
Envs that have essential roles in organismal development should evolve under continuous purifying selection. By contrast, those that inhibit replication of exogenous viruses may experience shorter-lived bouts of selection — when the exogenous virus becomes extinct or is replaced with a resistant variant, selection should be relaxed and the exapted gene subject to loss by drift57,144 (Fig. 1). Consistent with these predictions, syncytins have estimated ages ranging from approximately 12 million years to more than 80 million years7,89,90, whereas receptor-blocking Envs are younger, as reflected by narrower taxonomic distributions, insertional polymorphism and estimated integration times that are less than 20 million years ago57,96,145,146. Conceivably, many of the defective env sequences in the genomes of humans and other vertebrates may have once functioned to block viral entry but have since decayed owing to extinction of the selective agent57.
Exaptation of ERV non-coding elements
Integrated proviruses, and by extension ERVs, can alter the regulation of nearby genes12,147,148,149 and potentially influence the control of genes thousands of base pairs away150. Indeed, there are numerous examples of ERV LTRs functioning as novel promoters or transcription-factor-binding sites for genes, and there are now also examples of ERVs giving rise to novel regulatory long non-coding RNAs151,152,153. Several recent comprehensive reviews discuss the potential involvement of ERVs in both normal and aberrant gene regulation12,149,154. Importantly, thanks to ongoing acquisition and loss of ERV loci over evolutionary timescales, even closely related species vary in the composition and genomic distribution of ERV LTRs. Thus, through their effects on regulation of key genes, these elements may contribute to phenotypic diversification and, as a consequence, will be subject to exaptation by natural selection.
Recently, several lines of evidence suggest that ERVs may facilitate the concerted evolution of sets of genes that are regulated in coordination within so-called gene regulatory networks (GRNs)154,155,156,157,158,159,160. The coordinated regulation of genes can involve shared cis-acting regulatory elements (CREs), and the evolutionary rewiring of GRNs may be a source of phenotypic variation and species diversification161. At issue is whether shared CREs evolve de novo, which depends on random substitutions generating similar or identical motifs for multiple genes in a GRN, or whether there are mechanisms that facilitate concerted evolution of loci linked within GRNs162. Endogenization and parallel fixation of related ERV LTRs, containing similar or identical viral promoters and associated CREs, provide a compelling solution to the difficulties of the de novo hypothesis154,155.
Hypothetically, several unique properties of ERV could facilitate a role in GRN evolution. First, LTRs are densely packed with regulatory elements, including promoters and transcription-factor-binding sites (Fig. 2). These reflect the host range and tissue tropism of the virus at the time of integration, which may dictate the exaptive potential of any resulting ERVs. Second, although retroviral integration does not target specific motifs, it is also not perfectly random, with some retroviruses displaying preferences for transcriptional units or for promoter regions163. Thus, although endogenization may produce insertions distributed widely across the genome (and the host population), for some types of retrovirus, these insertions may be enriched in or near transcription units. Most of these are probably lost by drift or negative selection, but those that alter GRNs in beneficial ways will be favoured by natural selection. Third, sequence similarity within LTR families could facilitate the spread of a new motif in one locus to related loci, for example, by ectopic recombination or gene conversion59,164,165,166. Although speculative, a fourth feature of ERVs that may influence their role in GRN evolution in multiple ways is the propensity to form solo-LTRs. For example, solo-LTR formation would eliminate proviral sequences that are known targets for epigenetic silencing167 and may also activate the regulatory influence of the LTR on adjacent genes168. If solo-LTR formation is required to activate regulatory potential, then recombination and deletion would have to precede function, resulting in a temporal separation between the original integration event and the eventual manifestation of novel phenotypes subject to selection — possibly spanning hundreds or thousands of host generations. Moreover, solo-LTR formation may occur repeatedly at the same locus169, effectively increasing the probability of fixing a solo-LTR allele. Conversely, the probability of solo-LTR formation by homologous recombination decreases once the 5ʹ and 3ʹ LTR sequences begin to diverge170 such that the potential for exaptation may diminish with time.
At present, the most thoroughly documented ERVs are those of mammals, particularly those of mice and humans, although analyses of nonmammalian genomes are beginning to yield novel insights1,3,4,27,171,172. Molecular understanding of ERV biology, including viral functions, exapted cellular functions and contributions to disease, is even narrower, being mostly based on specific ERV or ERV families found in model organisms (for example, mice, chickens, livestock and pets). These are often inbred, domesticated species, which may not accurately reflect the process of endogenization as it occurs across generations in natural outbred populations. Broad comparative approaches may be the key to determining which biological properties, if any, predispose some retroviruses to germline invasion and for examining the impact of host biology and population dynamics on endogenization. Insights could come from studying natural populations currently in the early stages of endogenization55.
As a case in point, and despite the rapidly growing list of published examples, it is unclear whether LTR exaptation represents a major or minor mechanism of vertebrate GRN evolution173. To provide a major source of selectable variants, endogenization must produce many more insertions than are ultimately preserved by selection, yet little is known about the origins and initial population genetics of newly formed ERVs in natural populations. Consequently, incorporating LTR exaptation into general models of GRN evolution invokes several important questions: what triggers increases in ERV copy number (amplification bursts) in some lineages but not others? Have bursts of endogenization occurred with sufficient frequency during vertebrate evolution to explain the observed levels of diversity? Are these bursts temporally correlated with major speciation events or the appearance of novel phenotypes?
Similarly, the literature on exapted ERV proteins mostly relates to the identification and confirmation of cellular functions, with little attention given to understanding whether and how these genes undergo further modification for optimal function. Do they acquire additional regulatory refinements, and if they do, how? Do they experience additional adaptions in structure and function of the encoded proteins? Indeed, exapted ERV ORFs may prove generally useful for understanding how newly formed protein-coding genes gain interactions with other host factors and become integrated into existing regulatory circuits.
As a complement to molecular evolutionary analysis of ERVs, several new technologies now make it possible to test functional hypothesis directly. For example, deep-sequencing methodologies have been used for transcriptional profiling of ERV loci, for population-level analysis of germline integration and for detecting rare integration events45,46,54,55,63,174. Such approaches can be coupled with new techniques enabling analysis of individual ERV loci in primary cells and tissues and assessment of their regulatory potential. These include methods for identifying epigenetic modifications and DNA–nucleic acid interactions and protocols for analysing events at the single-cell level. As ERVs often belong to closely related, multilocus families, unambiguous assignment of sequencing reads to specific loci can be problematic, particularly when analysing younger, less divergent families. Thus, correlations between transcription and expression of ERV families and external triggers or various disease phenotypes have been observed, but such studies may lack the resolution to attribute observed biological effects to specific loci within a larger ERV family11. Useful insights come when care is taken to map reads precisely65,105,175 or to assess candidate ERV genes individually106. Finally, advances in genetic manipulation, including small interfering RNA and CRISPR–Cas, provide tools for perturbing and analysing native ERVs, including protocols for altering multiple loci in parallel at the cellular150 and organismal176 levels.
More than 100 years have passed since the discovery of the first retroviruses177,178, and a similar time span marks the origins of evolutionary genetics as a distinct discipline179. The study of endogenous retroviruses combines concepts from both fields, while the potential for ERVs to facilitate evolution of developmental and morphological diversity touches on fundamental questions in evolutionary developmental biology. The potential connections to cancer and autoimmune diseases have also drawn considerable interest from scientists in a variety of fields11. Going forward, ERV research encompassing any combination of these areas should be embedded in a framework of population genetics theory while incorporating knowledge and methods gained from over a century’s worth of research on all aspects of retrovirus biology.
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The author thanks J. Butler, B. Howell and the organizers of the 2018 Boston College Intersections Villa Faculty Writing Retreat for the opportunity to complete major portions of this manuscript; R. Gifford, L. Mulder and J. Henzy for helpful discussions; S. Whelan and V. Simon for providing offices for writing while on sabbatical leave at Harvard Medical School and the Icahn School of Medicine at Mount Sinai, respectively. Work in the author’s laboratory is supported by grants from the US National Institutes of Health (AI083118) and the US Department of Defense/Congressionally Directed Medical Research Programs (PR172274).
Nature Reviews Microbiology thanks A. Dupressoir, C. Feschotte, J. Frank and other anonymous reviewer(s) for their contribution to the peer review of this work.
The author declares no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- Endogenous retrovirus
(ERV). Heritable retrovirus-derived sequence elements found in the genomes of most or all vertebrates; ERVs usually originate as proviruses integrated into germline DNA.
Refers to the case when an allelic variant of a locus disappears from the population over time.
Refers to the case in which an allelic variant of a locus achieves a frequency of 100% in the population, thereby displacing all other alleles at that locus.
- Random genetic drift
Refers to the change in frequency of an allele over time owing to random chance (in the absence of selection).
- Long-terminal repeats
(LTRs). Direct identical repeats found at the 5ʹ and 3ʹ ends of a DNA provirus generated during reverse transcription of the retroviral RNA genome.
- CAAT box
A cis-acting transcription-factor-binding site frequently found upstream of eukaryotic promoters and in retroviral long-terminal repeats.
- Accessory genes
Viral genes that are dispensable for the essential steps of the viral replication cycle but that provide one or more functions that contribute to optimal viral fitness in vivo, such as antagonizing intrinsic and innate immune defences or modifying the metabolic state of the host cell.
Solitary long-terminal repeats (LTRs) lacking any other proviral sequence that usually arise by homologous recombination between the 5ʹ and 3ʹ LTRs of an ERV locus.
The amplification of a genomic DNA sequence by reverse transcription of an RNA intermediate followed by integration of the new DNA copies.
- Segmental duplications
Stretches of initially identical or nearly identical genomic sequences that arise by DNA duplication.
A trait that evolved on the basis of one function that has subsequently evolved to provide a different function.
- Superinfection interference
A phenomenon by which prior infection of a cell renders it resistant to reinfection by retroviruses using the same entry receptor; often mediated by the viral Env glycoprotein.
Glycoproteins of retroviral origin that fulfil cellular functions involving receptor-mediated membrane fusion; thus far, all reported syncytins function as placental syncytins.
A multinuclear layer that forms through fusion of mononuclear cytotrophoblasts.
- Restriction factors
Host-encoded factors that have evolved by natural selection to suppress or prevent viral replication at the cellular level.
- Purifying selection
A component of natural selection; refers to selection that eliminates deleterious or suboptimal variants of a gene or sequence that arise by mutation.
- R peptides
The last 17–20 residues of the cytoplasmic carboxyl termini of gammaretroviral Env proteins, which are cleaved off by the viral protease during virion maturation to activate fusogenic potential.
- ERV-L elements
An ancient family of related endogenous retrovirus (ERV) elements found in the genomes of all mammals; distantly related to spumaretroviruses.
- Exogenous virus
A horizontally transmitted virus, as distinguished from endogenous viruses.
- Positive selection
The selection that favours fixation of changes in a gene, such as when a virus escapes from virus-specific antibodies through changes in a target epitope.
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Johnson, W.E. Origins and evolutionary consequences of ancient endogenous retroviruses. Nat Rev Microbiol 17, 355–370 (2019). https://doi.org/10.1038/s41579-019-0189-2
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