Endogenous retroviruses: acquisition, amplification and taming of genome invaders

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ScienceDirect Endogenous retroviruses: acquisition, amplification and taming of genome invaders Marie Dewannieux1,2 and Thierry Heidmann1,2 Endogenous retroviruses are interspersed genomic elements that were generated after infectious retroviruses entered the germline of their host. They were initially identified as degenerate remnants of past infections, but new models of very recent or ongoing endogenisation are now emerging, allowing the real time investigation of the first steps of the coexistence between these elements and their host. Domestication of endogenous retroviruses involves several mechanisms, including transcriptional control of these elements and regulation of their mobility through the action of restriction factors. Recent studies also point towards an until-now unexpected role of the immune system for the control of these elements, even those that do not contain fully infectious copies. Addresses 1 CNRS UMR 8122, Institut Gustave Roussy, Villejuif, France 2 Universite´ Paris-Sud, Orsay, France Corresponding author: Heidmann, Thierry ([email protected])

Current Opinion in Virology 2013, 3:646–656 This review comes from a themed issue on Virus replication in animals and plants Edited by Ben Berkhout and Kuan-Teh Jeang For a complete overview see the Issue and the Editorial Available online 1st September 2013 1879-6257/$ – see front matter, # 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.coviro.2013.08.005

Introduction Eukaryotic genomes are filled with reiterated sequences that account for a major part of their DNA. Some fulfil a structural function in the organisation of the chromosomes and are grouped in a limited number of regions (e.g. telomeres, centromeres). But other are scattered all over the genome and are considered to be selfish mobile elements that increase their copy number by different strategies. Some replicate via a copy-and paste mechanism, using the enzymes they encode and DNA intermediates. Other elements consist of master copies that are transcribed by the cellular machinery and generate new copies via the retrotranscription of their RNA using a reverse-transcriptase (RT) that they either encode or borrow from other retroelements. In the human genome, the most successful retrotransposons consist of the LINEs and SINEs (long and short interspersed elements, respectively) whose main hallmark is a polyA tail present at their 30 end. The second category of retrotransposons are characterised by repeats present at both ends, the long Current Opinion in Virology 2013, 3:646–656

terminal repeats (LTRs). These LTRs surround open reading frames (ORFs) that encode structural and enzymatic proteins, including a RT. When RT domains are compared in phylogenetic analyses, the LTR elements cluster in the group of the mammalian infectious retroviruses, and are clearly distinct from the invertebrate LTR elements like copia or gypsy [1,2]. They are accordingly called endogenous retroviruses (ERVs).

ERV genomic sequences The complete sequencing of several mammalian genomes has allowed exhaustive studies on their ERV content. According to these data, ERVs account for approximately 10% of the genome. Some of these sequences correspond to solo LTRs, the products of homologous recombination events occurring between the two LTRs of an element which result in the excision of the internal ORF-containing part together with a LTR, while the other one is left in the genome as a fossil trace of the proviral insertion. This process is a simple, nonspecific way to delete ERVs using the cell machinery and limits their burden within the genome. Due to this mechanism, the elements still containing their internal sequence account for only 1% of the human genome, which nonetheless corresponds to thousands of proviruses. In most cases, the ORFs present between the LTRs are barely recognisable, being interrupted by frameshifts and premature stop codons. This degeneration is because of the accumulation of mutations with time, in the absence of any selective pressure for these ORFs to be conserved. When phylogenetic studies are performed on the retroviral sequences, they cluster in varying size groups of closely related elements (1–1000 copies per group, with up to 100 such groups per genome). Each of these groups corresponds to a family of ERVs whose members are all derived from the same remote ancestor. When placed in a tree together with present-day infectious retroviruses, the different ERV families are spread between the different types of retroviruses. Most of the early-identified ERVs were related to gammaretroviruses, but a significant subset of the ERVs cluster with the betaretroviruses. A few families are related to infectious spumaviruses (reviewed in [3] for human ERVs, see also [4–6] concerning recent examples in other species). No ERV related to the deltaretroviruses has yet been reported, and neither human or murine genomes possess endogenous lentiviral sequences, but other mammal genomes (from the rabbit and some lemurs) do [7,8], suggesting that all types of www.sciencedirect.com

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infectious retroviruses have the ability to become endogenised. Finally, in addition to this phylogenetic variability, the different families of ERVs also show a variety of structures: some of them contain ORFs homologous to the gag, pol and env genes of infectious retroviruses (and sometimes even encode accessory proteins, e.g. [5,7–9]), whereas others have completely lost their env gene. This is related to the amplification mechanism used by the family (see next section). There are also cases where the internal sequence is conserved within the family but is noncoding and has no homology with other ERVs (e.g. VL30, ETn elements in the mouse genome, reviewed in [10]). They correspond to parasitic elements that have evolved to hijack functional replicative ERVs, by encoding a viral RNA that can be packaged, reverse-transcribed and integrated by a heterologous element.

Life cycle of ERVs Colonisation of new genomes

The similarity between ERVs and their infectious counterparts led to the hypothesis that the former were generated when, in the course of a ‘normal’ infection, some viral particles gained access to the germline of their host where the viral genome integrated and has since then been vertically transmitted from one generation to the other (Figure 1). The structure and nature of the ERVs present in mammalian genomes indicate that, following the first integration, some elements are able to amplify within the genome and increase their copy number, but accumulate mutations at the same time. The high diversity observed among present-day ERVs indicates that this endogenisation process happens rather frequently. However, the situation observed now is the results of millions of years of co-evolution between the ERVs and their host. Most of the human elements for example were

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Mechanisms leading to endogenisation of retroviruses. ERVs originate from infectious retroviruses that can sometimes, in the course of a normal infection, reach the germline of the host where they integrate their genome as a provirus. The latter can then be transmitted vertically to the offspring, and also amplify with the genome, leading to the creation of a new family of ERVs. www.sciencedirect.com

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endogenised around 40 millions years ago (mya), including the most active human ERV family HERV-K (HML2) [11]. But even if most of the present day copies of the latter family are rather recent (estimated integration time: 0.1–5 mya), the effect of endogenisation cannot be deduced from their effect on the host since the virus has been in contact with its host for millions of year, and has had more than enough time to adapt to it. The study of retroviruses from sheep provided an example of recent endogenisation. enJSRV (endogenous Jaasiekte sheep retrovirus) is a family of endogenous betaretroviruses with around 30 copies in the sheep genome. They are closely related to the infectious JSRV, which is responsible for infectious lung cancer in animals. The first copies inserted around 5–7 mya in the genome of sheep ancestors, and new copies accumulated with time, with the latest identified estimated to be less than 200 year old, suggesting that some enJSRV are still active (reviewed in [12,13]). This constitutes a very good model to study the coevolution of an ERV with its progenitor (which is still infecting animals) and the host. The most striking example of ongoing endogenisation known to date in mammals is occurring in koalas and was reported in 2006 ([14], reviewed in [15]). A significant proportion of koalas are suffering from leukaemia and lymphosarcoma which are associated with the presence of gammaretroviral-like particles in the bone marrow, and accordingly with a retrovirus called KoRV (koala retrovirus) which was thought to be endogenous owing to its presence in the DNA of all tissues analysed [16]. Tarlington et al. [14] confirmed its endogenous status in animals from Northern Australia, with the demonstration of proviruses being transmitted vertically like mendelian alleles and present as DNA in the sperm of infected animals. The insertion pattern is compatible with a very recent endogenisation, with most of the proviral loci still not fixed. The very high viral load observed in the plasma of infected animals is also suggestive of a recent event since ERVs are usually silenced in normal tissues. More recently, a study confirmed these data for koalas from Northern Australia, with all tested animals positive for KoRV DNA with a high copy number (approx. 150 per genome), consistent with an endogenous retrovirus [17]. However, in animals originating from Southern Australia, the prevalence was lower, as was the copy number (1.5– 0.0001 provirus per genome), suggesting an ‘exogenous’, infectious virus. In addition, the successive studies performed suggest a tendency towards an increase in the prevalence of KoRV in these regions, which is also in agreement with what was inferred from the analysis of old koala samples conserved in museums [18]. All these data together suggest that we are observing in real time the propagation and concomitant endogenisation of this retrovirus. Current Opinion in Virology 2013, 3:646–656

Following the initial description of this model, KoRV is being better characterised. It is closely related to GaLV (gibbon ape leukaemia virus), a retrovirus isolated in a couple of captured gibbon apes, and also to an endogenous retrovirus found in the Asian mouse Mus caroli (reviewed in [19]). Comparison of the properties of GaLV and KoRV in cultured cells suggested that adaptations have already occurred, with mutations being found that significantly decrease the infectivity of KoRV and could represent the first steps towards the domestication of the virus by its new host [20]. Amplification of ERVs within genomes Mechanisms of amplification

Most ERV families in mammals contain multiple copies that derive from one another after the initial infection of the germline. Different studies showed that the amplification mechanism used by a family is directly related to its structure: in ex vivo experiments, functional autonomous ERVs possessing an env gene depend on it for their amplification, which also requires the expression of a functional receptor, suggestive of an amplification occurring through successive extracellular re-infections of the cells [21,22] (Figure 2). This is also in agreement with an in silico study performed on the human HERV-K (HML2) elements which showed that the envelope gene has been under the same purifying selection pressure as that of the other retroviral genes during their amplification in primates [23]. Conversely, ERVs devoid of an envelope gene encode retroviral particles that stay within the cells and replicate using a strictly intracellular pathway [24–26] (Figure 2). Experimentally providing them with a heterologous envelope protein rescues neither the localisation of the particles that remain confined inside the cells, nor their ability to perform extracellular infectious cycles [22,26]. A tendency towards intracellularisation

In the mouse genome, ERV family size goes from a few elements to more than 1000. Rather strikingly, it seems that the more successful families with regards to the copy number correspond mainly to elements devoid of an envelope gene. This is also consistent with their observed insertional mutagenesis activity: the envelope-less IAP elements are thought to be responsible for approximately 10% of all de novo mutations, and the ETn elements, which are another important source of insertional mutagenesis in vivo, are mobilised by env-less MusD elements [25]. A more global in silico analysis extended this observation to other mammalian genomes by showing that envless ERVs amplify to much higher copy number than those with an env gene [27]. Many reasons can account for a better efficiency of the envelope-less ERVs. First, strictly intracellular elements keep their encoded particles concentrated within the cell instead of having them diluted in the extracellular space, www.sciencedirect.com

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Figure 2

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Comparison of the replicative cycles of ERVs with an extracellular (top) or strictly intracellular (bottom) pathway. Top: The elements with an extracellular phase (blue) are transcribed and translated into proteins that assemble into viral particles. The latter bud from the cell membrane, initiating the extracellular phase of the element, and can thereafter infect another cell after interacting with their cellular receptor (schematised in orange). The viral capsid is then released into the cytoplasm where reverse-transcription of the viral genome can occur, leading subsequently to the insertion of a new provirus into the genome. Bottom: As compared to elements with an extracellular phase, ERVs with a strictly intracellular cycle (in green) have a Gag protein with a different targeting signal, and no (or a non-functional) env gene (schematised by a black cross in place of env on the green proviruses). These elements are also transcribed and translated, leading to the formation of intracellular particles. They can either be assembled directly into the cytoplasm, as represented on the figure, or bud into an intracellular compartment (e.g. endoplasmic reticulum for IAP or ERV-L elements, not represented here). Reverse-transcription occurs within the particle, and the resulting provirus can be inserted into the genome of the same cell.

which increases the probability of insertion of a new element in the genome. A local concentration of particles is also certainly an asset against the host restriction factors, which are saturable: intracellularly sequestered particles could possibly accumulate and finally overcome the endogenous pool of a given restriction factor, which is much less likely to occur if the particles come from outside the cells. In addition to the dilution effect, secreted particles can also become the target of the immune system and be destroyed before they have a chance to reach an appropriate target cell. Two independent experimental models have shown that the transition between a strictly intracellular transposon and an infectious LTR element can be relatively simple. MusD elements are env-less ERVs which closest relative is MPMV (Mason-Pfizer monkey virus). They encode strictly intracellular particles that accumulate within the cytoplasm. This intracellular transposon can be induced to produce infectious extracellular particles simply by readdressing Gag to the cell membrane through the addition of MPMV myristilation signal and providing a www.sciencedirect.com

heterologous envelope protein, leading to a loss of its transposition ability [26]. The study of IAP/IAPE elements proved this was not simply a molecular biologist trick and that it indeed happened in vivo. IAP and IAPE are two closely related ERV families, with the former having lost most of env in the course of evolution. The only other difference in their coding regions corresponds to the N-terminal end of the structural Gag protein: in IAPEs, it is targeted to the cell membrane, whereas in IAPs particles are addressed to the cisterns of endoplasmic reticulum. Swapping this region between the two elements is enough to exchange the localisation of the corresponding particles as well as the amplification mechanism (from intracellular transposition to extracellular infection, and reciprocally), provided that they are complemented with an envelope protein when required [22]. This suggests that mice were first colonised by the IAPE family. During its amplification through re-infection, some of its copies evolved, somehow getting a new Gag addressing signal and losing env, and turned into ‘intracellularised’, highly efficient transposons that spread at a high copy number within the genome. Current Opinion in Virology 2013, 3:646–656

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So the transition from an infectious cycle to an intracellular retrotransposition mechanism is relatively simple, and increases the amplification efficiency of the element, suggesting it could (or should) be an evolutionary trend for ERVs. However, the gain in transposition efficiency comes at a cost for the element: it becomes strictly dependent on its host and its replication machinery for its long-term survival since the loss of its infectious properties prevents further genome colonisation by horizontal transfer.

Figure 3

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G9a

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CH3

The mechanisms through which ERVs are silenced have been well studied over the years, and it was showed that this regulation is controlled mainly via chromatin structure (e.g. DNA and histone methylation, histone deacetylation). A number of genes are involved in controlling ERVs (see Figure 3), including DNA methyltranferases, with Dnmt1 being responsible for the maintenance of the methylation pattern [28,29], whereas Dnmt3a, Dnmt3b and Dnmt3L are in charge of the establishment of the de novo methylation pattern during development [30,31]. To be active, these proteins depend on the presence of Lsh, a helicase of the SNF2 family [32–34]. Other proteins have been involved in ERVs regulation, including Np95 (Nuclear protein of 95 kDa) which interacts with Dnmt1, Dnmt3a and Dnmt3b [35,36], and G9a, a H3K9 histone methyltransferase whose presence is required for the establishment of ERV methylation [37,38]. Another pathway goes through ESET (also called SETDB1 or KMT1E) [39], another H3K9 Current Opinion in Virology 2013, 3:646–656

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ERVs are generally not expressed in normal conditions, including in most mammalian cell lines. However, they can be induced, in particular when the cells are under stress or by using chemical treatments. The latter act by modifying the structure of the chromatin (e.g. inhibition of DNA methylation or of histone deacetylation). This indicates that ERVs still possess the capacity to be expressed but are, in normal conditions, actively repressed by the cellular machinery.

+ Dnmt3a Dnmt3b Dnmt3L

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Multiple mechanisms of ERV taming by mammals As ERVs are present at high copy number and can amplify, they represent a major risk for genome stability and survival of the host. However, in crucial conditions, their properties also mean they can be seen as a potential source of innovation that can be used for modifying and re-shuffling the genome. It is thus important for the host to be able to ‘tame’ these elements, by restricting their activity in usual conditions while still keeping ways to activate them. This taming operates both through regulation of their expression and functional blockages, using either genome-encoded so-called restriction factors that directly interfere with the retroviral replicative cycle or even the immune system.

Np95

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Proteins involved in ERV transcriptional regulation. (a) A first level of control consists of cytosine methylation of the DNA. The key enzymes for this reaction are the DNA methyltransferases, with Dnmt1 being responsible for maintenance of methylation (i.e. methylation of hemimethylated DNA after DNA replication) whereas Dnmt3a, Dnmt3b and Dnmt3L (a catalytically inactive cofactor) cooperate to achieve de novo methylation. The DNA methyltransferases are associated with G9a, a H3K9 methyltranferase, and Np95, both of which are necessary for a proper methylation of ERVs. The latter also depends on the presence of Lsh, a DNA helicase. In the case of de novo methylation, the mechanisms responsible for the targeting of the sequences to be methylated are unknown. (b) A second pathway of regulation depends on TRIM28. TRIM28 is targeted to ERV sequences by members of the KRAB-ZFP family that bind to a specific PBS. Transcriptional repression is achieved by a complex of proteins, including ESET, a H3K9 methylase, HP1 and the NuRD complex responsible for histone deacetylation.

methyltransferase that represses ERVs in cooperation with TRIM28 (also called KAP1: Kru¨ppel-associated box (KRAB) associated protein 1) [40,41]. More details can be found in recent reviews (e.g. [42]). All the mechanisms described above are rather general, and the enzymes involved cannot account for the specific targeting of ERVs. In the silencing complex consisting of TRIM28, ESET, NuRD (nucleosome remodelling and www.sciencedirect.com

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deacetylase) complex and HP1, the specificity is provided by another partner, a member of the large family of KRAB domain zinc finger proteins (KRAB-ZFPs) that can bind DNA. ZFP809 is a member of this family and targets ERVs (and infectious proviruses) related to murine leukaemia virus (MLV) through their conserved 18nt primer binding site (PBS) [43]. The PBS of other retroviruses can also be silenced in mouse ES cells through TRIM28 [44], and this is likely performed through other KRABZFPs, suggesting they could silence a wide variety of ERVs. Another mechanism explaining how ERVs can be selectively silenced resides in piRNAs. These are short (24– 30 nt) RNAs that are either encoded by long genomic clusters that are processed after transcription (primary piRNAs) or generated from mRNA during a so-called ping-pong amplification mechanism (secondary piRNAs). piRNAs are associated with proteins of the Piwi family [45] and provide them with their sequence specificity. In mice, both MIWI2 and MILI piwi proteins are involved in ERV silencing [46,47]. The mechanism through which they act is not fully understood, but it involves targeted degradation of ERV mRNAs by antisense piRNAs as well as ERV methylation [48,49], suggesting that the piRNAs serve as guides used to target genomic sequences to be silenced. The complete piRNA pathway has also been

reported to involve the MOV10L1 RNA helicase [50], and Tudor domain-containing proteins, some of which can specifically interact with MILI or MIWI2 ([51], reviewed in [52]) and form, together with other proteins, cytoplasmic granules (pi-bodies and piP-bodies respectively) involved in mRNA degradation and transcriptional repression [53]. More details can be found in recent reviews (e.g. [54]). Regulation by restriction factors

It has long been known that animals or cell lines can express proteins, called restriction factors, able to specifically inhibit infection by various retroviruses. Since infectious and ERVs share similar replication cycles, these factors can, in theory, interfere with both types of viruses, and even possibly determine which can be endogenised. Restriction factors act at different steps of the retroviral cycle (Figure 4). Fv4 is a mouse gene derived from the envelope of an endogenous ecotropic MLV provirus. It protects cells from infection by related viruses by interfering with the proper membrane expression of their shared receptor, a usual mechanism called receptor interference [55]. It also limits the production of viral particles in infected cells through interactions with the virus envelope proteins that render them non-functional [56]. This gene

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Restriction factors act at different steps of the replicative cycle. The replicative cycles of ERVs (with either an extracellular or intracellular pathway) are schematised. The steps at which restriction factors can act are indicated in red. Some of them are rather general, and are effective against a wide variety of elements (e.g. APOBEC3, tetherin, proteins acting on the chromatin structure), whereas others are specific to a given ERV family (e.g. Fv4 against ecotropic MLVs, Fv1 against MLVs, enJSRV Gag* and enJSRV Env against exogenous strains of JSRV). enJSRV Gag* refers to endogenous JSRV Gag variants that are effective against other endogenous and exogenous JSRV elements. www.sciencedirect.com

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can thus play a role both by limiting the amplification of already endogenised ecotropic MLVs, and by protecting the host from exogenous ones. Fv1 is another murine restriction factor. It is related to the gag gene of the spumaretroviral ERV-L family [57,58] and targets MLVs in vivo. It blocks the infection cycle after the reverse-transcription step, but before integration of the cDNA into the cell genome [59]. However its mechanism of action is not known. This gene exists under several allelic forms in mice, with different targets, and is responsible for the varying sensitivity of mouse strains against different isolates of infectious MLVs. Emergence of infectious retroviruses following recombination of endogenous ERVs is quite common in mice. In several independent cases, a key step in the recombination process involved the region of the gag gene targeted by the Fv1 restriction factor, transforming the originally restricted virus in a resistant one in the context of its host (e.g. [60,61,62]). This strongly suggests that Fv1 plays a role in the control of endogenous MLVs and their replication. Sheep enJSRVs also contribute restriction factors against their exogenous counterpart. Some env ORFs limit infection through receptor interference [63]. Two enJSRV copies also possess mutated gag genes encoding dominant negative proteins that interfere with the viral cycle of JSRV (both exo and endo-genous) at a late stage ([64], reviewed in [13]). Interestingly, the most recently integrated enJSRV has been found to harbour a single point mutation within Env that makes it resistant to the late restriction mediated by the endogenous Gag [65]. These mutated enJSRV ORFs indeed act as restriction factors, but are not yet fixed in the population. They are good examples of the successive adaptive mutations that occur in a virus and its host to counteract each other. Study of HIV-1 led to the identification of another restriction factor, APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypetide-like 3G), a member of a multigenic family of proteins present in mammals ([66], reviewed in [67]). The number of APOBEC genes varies between species, with some of them very conserved like APOBEC1 or AID (Activation-Induced Deaminase). ABOBEC3 is less conserved between species, with the human lineage having experienced multiple gene duplications that generated 7 paralogs. APOBEC3G and other members of its subfamily restrict retroviruses by being incorporated in the budding viral particles. When they infect a new cell, APOBEC3 causes dC to dU mutations at specific sites of the newly formed viral ssDNA during reverse-transcription, ending with the integration of a mutated, defective version of the viral genome ([68– 71], reviewed in [67]). Ex vivo experiments showed that each ERV studied could be restricted by at least one APOBEC3 gene [72–76]. Even more interestingly, in Current Opinion in Virology 2013, 3:646–656

silico analyses of ERV families showed that some individual proviruses contained distinctive APOBEC3 induced mutations, indicating that these proteins were active against them at the time of their amplification and may have contributed to their inactivation [72,75,77–79]. However, it seems that at least some of them have evolved resistance mechanisms (as HIV-1 does through the vif protein [66], or FFV (feline foamy virus) through bet [80]), since it was reported that some MLV-encoded glycosylated Gag proteins can protect their virus from mouse APOBEC3 mediated restriction [81,82]. Another protein, TRIM5a, has been identified as a restriction factor present in most mammals [83], with some primates having instead the related TRIMCYP gene (reviewed in [84]). The varying forms studied have different specificities, with some active against HIV1, SIV or MLVs. They block early in the retroviral lifecycle, after the entry of the particle but before the reverse-transcription takes place (reviewed in [85,86]). The effect of TRIM5a on ERVs has not been extensively investigated, but it was shown that a reconstructed HERV-K element is restricted by neither human and rhesus macaque TRIM5a nor owl monkey TRIMCYP [87]. This is in agreement with another study indicating that HERV-K is still likely to actively amplify in humans [88], which could not occur if it were restricted by human TRIM5a. According to another study, CERV1/PtERV1, a reconstructed ancestral endogenous retrovirus from the chimpanzee genome, is sensitive to human TRIM5a, which could explain why this element is absent from the human genome [89]. However this result could not be confirmed by another group [77], and the controversy has not been solved. More recently another antiretroviral restriction factor, Tetherin, was identified. This protein keeps fully assembled viral particles attached to the producer cell and thus prevents their release into the surrounding space ([90], reviewed in [91,92]). Its role was initially demonstrated on HIV-1 particles, but it is active against all retroviruses investigated to date (as well as other enveloped viruses), and thus it is very likely it can restrict most ERVs (or at least those with an extracellular cycle), even if this has only been properly demonstrated for a reconstructed HERV-K core [93] and enJSRV elements [94]. MOV10 is another protein that was first investigated for its role in HIV-1 replication [95–97] and was thereafter found to also act against ERVs. It is a RNA helicase which is packaged into HIV-1 virions and interferes with reverse-transcription. There is a general agreement on the fact that its overexpression leads to a decrease in infectious viral particles production, whereas effects of its downregulation are not as clear (discussed in [98]). MOV10 overexpression also leads to a decrease in IAP transposition, but the effect of its silencing is also a www.sciencedirect.com

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subject of controversy, and may depend on the cell line used [98,99]. Interestingly, MOV10 is also active against LINEs and SINEs transposition [100]. Therefore it seems to act as a general regulator of retrotransposon activity in mammals.

linked to a lack of anti-ERV antibodies. These two papers clearly demonstrate that the immune system plays an important role in the control of ERV and prevents reactivation of theoretically defective families.

Conclusion All these restriction factors are effective against some, but not all viruses, independently of their origin. Indeed, thanks to their usually high mutation rate, the latter can evolve and adapt so as to avoid restriction by their host (cf. the previously mentioned recombination of endogenous MLVs that affect their restriction by Fv1). In this respect, the concomitant presence of numerous infectious and endogenous retroviruses may represent opposite evolutionary forces at play on these restriction factors, limiting their efficiency and driving their positive selection. Regulation by the immune system

In mammals, infectious viruses are mostly controlled by the immune system. Owing to the similarities between ERVs and infectious retroviruses, it is very likely that the former are subjected to the same control. Indeed, as already described, some ERVs are under the control of the restriction factors, which are often regulated by type I interferons (IFN) (e.g. tetherin, APOBEC3, TRIM5a), and can be as such considered as part of the innate immune system. In addition, some of these restriction factors (e.g. tetherin, TRIM5a) act also as pathogens sensors [101–103]. Activation of one by an ERV is susceptible to induce the expression of others, and thus lead to a coordinated response and a better control of the element. Another IFN-regulated gene, which is not considered as a restriction factor, has also been involved in the control of ERV activity. It is Trex1, which encodes a cytoplasmic 30 ! 50 DNA exonuclase. In Trex1 deficient cells, ERVderived single strand DNA accumulates and, conversely, overexpression of Trex1 decreases the transposition efficiency of transfected ERVs, probably through the degradation of newly synthesised ERV DNAs [104]. Two recent studies have in addition shown the involvement of the adaptative immune system in ERV regulation. In one case, the authors showed that disrupting antibody production (through different gene modifications) in C57BL/6 mice leads to increased expression of ecotropic MLVs, and eventually to the resuscitation of infectious elements through recombination of the activated loci, responsible for lymphoma in the animals [62]. In the other study, the authors achieved induction and recombination of the same ecotropic ERVs through inactivation of Toll Like Receptor 7 (TLR-7) in C57BL/6 mice, leading to highly viremic animals that developed leukaemia when TLR-3 and TLR-9 were additionally inactivated [105]. In the latter case the viraemia was also www.sciencedirect.com

In this review, we went over the main aspects of ERV biology, especially in regard to their amplification within genomes and the way they are tentatively tamed. However, from the day they are endogenised, ERVs become part of the genome and can be, given time, co-opted to serve their new host’s interests. This was briefly mentioned in this review since some of the restriction factors themselves were initially ERV genes whose properties have been altered, turning them into antiviral mechanisms. Co-option of endogenous retroviral env genes has also happened repeatedly in mammalian evolution to serve another function, namely development of the placenta, where the fusogenic properties of the envelope proteins have been enhanced and put to work for the building of a syncytial layer that both acts as a barrier and promotes efficient exchanges between the mother and foetuses ([106], reviewed in [107]). Other examples of co-option are due not to ERV encoded ORFs, but rather to their transcriptional regulatory abilities (reviewed in [108]), with several cases having been described where LTRs alter the expression pattern of their insertion site, or where a family of elements are used to synchronise the expression of their surrounding genes [109,110]. Altogether, ERVs are inescapable inhabitants of eukaryotic genomes involved in reciprocal, controlled, and in some cases even symbiotic interactions with their host.

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