Endogenized viral sequences in mammals

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ScienceDirect Endogenized viral sequences in mammals Nicholas F Parrish1 and Keizo Tomonaga2,3,4 Reverse-transcribed RNA molecules compose a significant portion of the human genome. Many of these RNA molecules were retrovirus genomes either infecting germline cells or having done so in a previous generation but retaining transcriptional activity. This mechanism itself accounts for a quarter of the genomic sequence information of mammals for which there is data. We understand relatively little about the causes and consequences of retroviral endogenization. This review highlights functions ascribed to sequences of viral origin endogenized into mammalian genomes and suggests some of the most pressing questions raised by these observations. Addresses 1 Section of Surgical Sciences, Vanderbilt University Medical Center, Nashville, TN 37232, United States 2 Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan 3 Department of Tumor Viruses, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan 4 Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto University, Kyoto 606-8507, Japan Corresponding authors: Parrish, Nicholas F. ([email protected]) and Tomonaga, Keizo ([email protected])

Current Opinion in Microbiology 2016, 31:176–183 This review comes from a themed issue on Megaviromes Edited by Didier Raoult and Jonatas Abrahao

http://dx.doi.org/10.1016/j.mib.2016.03.002 1369-5274/# 2016 Elsevier Ltd. All rights reserved.

Introduction Taxonomists distinguish between exogenous retroviruses (Figure 1a) [1], retrovirus-like endogenous retroviruses [2], retrotransposons that contain long-terminal repeats (LTRs) as do retroviruses [3], and autonomously-mobile retrotransposons lacking LTRs [4–6] all of which are mobile nucleic acid elements encoding reverse transcriptase and inferred to have a common origin on the basis of phylogenetic analysis of this protein. It has been argued that an infectious retrovirus was ancestral to the elements now comprising each of these groups [7]. Some members of each group move via an RNA intermediate in mammalian germlines, that is to say, vertically. Cross-individual or cross-species horizontal mobility is a defining feature of exogenous retroviruses, but has also been observed for endogenous Current Opinion in Microbiology 2016, 31:176–183

retroviruses [8–11], LTR retrotransposons [12–14], and autonomous non-LTR retrotransposons [15,16]. Recent large-scale genome analyses provide evidence for considerable host-switching by endogenous retroviruses, an observation consistent with extensive yet-to-be discovered exogenous retroviral diversity [17,18,19]. The boundaries between taxonomically distinct groups of retroelements are predicated on our cross-sectional perspective with respect to time and the current limited depth of sampling; nevertheless, we have four examples in which a host species is infected by a virus straddling the exogenous/endogenous retrovirus divide: clearly koala [20] and likely sheep [21], opossum [22], and polar bear [23]. It can be expected that the great cloud of mobile retroelement diversity will continue to expand and merge as more data is collected and analyzed. The important realization that these elements have contributed vast swaths of mammalian genomes is ongoing. Conservative estimates that 40% of the human genome is reverse transcript in origin [24] balloon when considering that nearly 80% of the genome can be identified as repeat-derived using algorithms with increased sensitivity for short and degraded sequences [25]. Given their massive contribution to the overall content of the genome, in comparison to the exome there disproportionately little evidence that sequences derived via reverse-transcription of non-self RNA contribute to mammalian organismal physiology. This reflects the nature of scientific change from a historically entrenched paradigm. Prior to the genomic era, selective breeding experiments allowed generation of chicken and mice lacking specific endogenous viral elements [26–28]. At this time, the concept of even one endogenous provirus was very recently a controversial tenet [29]. Perhaps thusly, the possibility that the endogenous viral sequences in question represented the tip of a deep iceberg was neglected; these experiments were interpreted to suggest that endogenous viral elements were superfluous to normal development [26]. Despite the recognition of Barbara McClintock’s foresight into the importance of transposons as ‘controlling elements’ [30], it has taken a long time for comprehensive alternative paradigms to be presented in rigorous forums [31].

Endogenous retroelements in health and disease Perhaps the most frequently cited example of retro-endogenization playing an important role in mammalian physiology comes from the exaptation of retroviral envelope genes encoding syncytin proteins, which play a role in cell– cell fusion during placentation (Figure 1b) [32,33]. In humans, two endogenous retroviral envelope genes are www.sciencedirect.com

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Figure 1

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(a) Retroviral to endogenous retrovirus lifecycle. Upon engagement of host receptor (i), the viral payload enters the cell. The viral RNA genome is uncovered by the structural proteins protecting it (ii) and, at some point, reverse transcribed into viral cDNA (iii). Host pattern recognition receptors (PRRs) may recognize viral RNA or cDNA and activate signaling cascades that lead to abortive infection (iv). Alternatively, the viral cDNA may be integrated as a proviral copy with the host chromosome (v) from which progeny virus are made (vi). Upon integration of a retrovirus into a germ cell (vii), a provirus may be horizontally transmitted via the germline as an endogenous retrovirus. As an endogenous retrovirus or retroelement, further propagation within the germline is possible in the absence of infectious virion production (viii). (b) Syncytin-mediated fusion and immune suppression. Expression of fusion-competent endogenous retroviral Envelope glycoproteins (Env) on the surface of trophoblast cells of the placenta enables production of multinucleated syncitiatrophoblasts (i). Via an unknown mechanism, syncytins have been proposed to prevent recognition of fetal cells as foreign by the allogeneic maternal immune system (ii). (c) Receptor interference. Expression of endogenous Env interacts with the receptor of closely related exogenous viruses, either in a cell-associated (i) or cell-free (ii) form, leading to either degradation (iii) of the receptor or competitive inhibition preventing efficient interaction of exogenous virus with the host cell receptor. (d) Dominant-negative protein production. After infection of a cell with an exogenous virus (i) which is capable of producing an endogenous viral structural protein (ii), formation of a functional group antigen (Gag) lattice is interrupted (iii) leading to production of non-infectious virions (iv). (e) Innate immune modulation. Environmentally triggered or stochastic expression of endogenous retroelement RNA or reverse transcription of this RNA in to cDNA (i) generates a substrate recognized by PRRs (ii) which transduce a signal to the nucleus (iii) activating antiviral gene transcription programs and leading to a multi-effector antiviral state (iv). (f) Adaptive immune modulation. Activation of PRRs by endogenous triggers (i) is required for expression of anticipatory receptors, antibodies (ii), in T-independent B cell responses. Endogenous retroelement reverse transcriptase (RT) is required for reverse transcription (iii) and integration (iv) of sequences from viruses that do not encode reverse transcriptase activity. These non-retroviral endogenous viral elements may then produce dominant negative viral proteins (see d) or small RNA (v) capable of base-pairing with exogenous viral RNA, leading to viral gene silencing (vi). (g) Pluripotency. Endogenous retrovirus transcripts may act as nuclear non-coding RNA (i) or may encode proteins that bind to host RNAs involved in pluripotency (ii). Germ cells with overactive endogenous retroelements (iii) may die prior to zygote formation (iv), allowing propagation of only those gametes with relative genomic integrity.

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expressed specifically on trophoblasts, the cells that fuse to form syncytia critical to establishing the materno-fetal interface. Interestingly, in mice two endogenous retroviruses integrated into different genomic positions relative to those encoding human syncytins (i.e. the viral integrations are not orthologous, not related by descent) have been implicated in this process and are activated by a transcription factor shared in common by humans and mice [34]; the genetic absence of these syncytins in mice results in abnormal placental syncycia formation [35,36]. Across the Therian subclass, there have been at least ten independent retroviral integration events from which the envelope gene has been convergently exapted to function as a syncytin [37]. In addition to their proposed role in cell–cell fusion, transduction of some syncytins allows engraftment of otherwise rejected allogeneic tumor cells, presumably by suppressing immune recognition [38]. Viral modulation of cell surface proteins involved in immunity, including MHC molecules, is common [39,40], raising the question of whether or not this is the case for syncytins. In any case, expression of endogenous viral proteins in the placenta may have both anatomic and immunological consequences. Prior to their implication in placental immunomodulation, endogenous viral sequences were shown to mediate immunity to closely related exogenous viruses. The bestdescribed mechanism has been termed receptor interference (Figure 1c), whereby an endogenous retroviral envelope glycoprotein (Env) binds to and sequesters the receptor of the very closely related exogenous virus. This has been shown for endogenous Jaagsiekte sheep retrovirus (enJSRV) [41], a provirus related to murine leukemia virus (MLV) [42,43], and endogenous feline leukemia virus [44]. Mtv-3, a provirus related to murine mammary tumor virus (MMTV), was shown to correlate with resistance to MMTV through an as yet unknown mechanism [45]. Not only Env, but other endogenous viral proteins can exert a dominant-negative role on infection by related viruses. This has been shown for enJSRV group antigen [46] proteins [47] as well as for the Gag encoded by Fv1, identified as a murine leukemia virus restriction factor (Figure 1d) [48]. Interestingly Fv1 is itself not closely related to MLV at the nucleotide level, and while the first identified phenotype was resistance to this betaretrovirus, alleles of this gene in various mouse species have recently been shown to restrict infection by a lentivirus and a spumavirus [49]. Thus while initially considered to confer type-specific immunity to very closely related exogenous viruses, in some cases endogenous retroviral protein expression can have a broader antiviral effect. Sequences derived from endogenous retroelements may play a physiological role in immunity via activating innate immune pathways when transcribed to RNA or upon reverse transcription to cDNA. Cells treated with the Current Opinion in Microbiology 2016, 31:176–183

DNA demethylating agent 5-aza-20 -deoxycytidine massively increase expression of endogenous retrovirus and other retroelement RNA and this correlates with interferon-stimulated gene response, however only in cells lacking functional alleles of p53, one of the most frequently mutated tumor suppressor genes in human neoplasms [50]. Several recent papers have extended [51] and clarified these observations in the case of colorectal cancer [52] and ovarian cancer [53]. Retroelement RNA or cDNA has also been suggested to act in a similar mechanism in non-transformed cells (reviewed in [54,55]). Additionally, in germ cells in the mouse testis, expression of the autonomous non-LTR retrotransposon LINE-1 induces expression of interferon beta, especially when the endonuclease domain of the polymerase gene is functional [56]. Moreover, release of retroelement RNA bound to the Ro60 autoantigen increased interferon-regulated gene stimulation [57]. Together these data support a role for endogenous retroelement sequences, generally acting at the RNA or cDNA level to trigger pattern recognition receptors which have previously been implicated in recognizing exogenous viruses, in modulating innate immunity (Figure 1e). For each physiologic function with a hypothetical survival benefit that a retroelement might play, it follows that a corresponding pathology results from improper control of the retroelement. The full literature associating human endogenous retroviruses with disease is beyond the scope of this review (see [58]), but there is growing evidence that a subset of autoimmune diseases with increased type 1 interferon signatures may, etiologically, represent syndromes of retroelement dysregulation [54,59,60]. In addition to innate immunity, endogenous retroviruses have been proposed to play a role in adaptive immunity. Endogenous retrovirus transcript/reverse-transcript sensing as described above is required for generation of some T cell-independent B cell immune responses; preventing cDNA formation by these retroelements with reverse transcriptase inhibitors has been shown to block antibody formation (Figure 1f) [61]. This data is a striking realization of a hypothesis presented over 35 years ago [62]. In addition to a direct involvement, several investigators have proposed that endogenous retrovirus sequences present in major histocompatibility gene families are important for generation of diversity in these genes, which are critical for adaptive immunity [63–65]. A role for endogenous retroviruses in generation of diversity at antibody variable segment gene loci has also been proposed [66]. Finally, endogenous retroelements are required for endogenization of viruses which do not encode a reverse transcriptase, a recently described phenomenon [67] with a surprising multitude of examples [68]. Endogenization of such sequences may play a role in typespecific immunity via dominant negative protein production as described above [69] and/or via an RNA silencing mechanism analogous to CRISPR/Cas [70]. www.sciencedirect.com

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Endogenous retrovirus expression is high in germ cells, preimplantation embryos and the placenta [26,71,72]. This led to the hypothesis that they play a role in early human development [73–75], which has been born out at least in part with the evidence relating to syncytins. However, as gene expression in some of these tissues is pervasive [76], it was not clear that increased expression of endogenous retroviral sequences in pluripotent tissues implied function. Surprisingly, recent data suggests that endogenous retroviral expression is not only associated with pluripotent tissues but in fact appears to play a role in establishing or maintaining the pluripotency phenotype itself [77–79]. One model suggests that the endogenous retroviral RNA acts as a nuclear non-coding RNA scaffold to activate transcription of specific genes (Figure 1g) [80,81]. Another non-mutually exclusive model proposes that proteins expressed from endogenous retroviral transcripts could induce antiviral proteins and influence translation of subsets of cellular RNAs [82]. Mirroring the multiple independent syncytin exaptations across placental mammals [37], it is remarkable that a physiologic function as basal as maintenance of pluripotency is critically influenced by endogenous sequences that entered primate genomes by some estimates as recently as 40 000 years ago [83]; if endogenous retroviruses are responsible for this function in other mammals, as seems the case at least in mice [84], they are different viruses. The maintenance of totipotency across generations, the role of the germline, is also influenced by retroelements. Most mouse oocytes die prior to ovulation and recent work has suggested that variable levels of LINE-1 activity underlie this attrition, supporting a model in which the eggs with optimal control of this potential mutagen are selected for potential fertilization [85]. As similar mechanisms may be in play in spermatogenesis [56,86] and even Caenorhabditis elegans germ cell development [87], this may also be broadly conserved in principle yet with potentially different retroelement actors in each setting. As expected for nuclear elements with a role maintaining a state of unlimited cell division, dysregulation of retroelements has been proposed to be involved in carcinogenesis [88–92]. The recent data relating to hepatocellular carcinoma (HCC) demonstrates several of the diverse mechanisms involved: somatic integrations of LINE-1 in some cancers drives oncogene expression [93], integration of a gene from the non-retroviral hepatitis B virus (HBV) gene into the human genome creates a chimeric transcript with LINE-1 which functions as a long non-coding RNA in as many as 20% of HBV-associated tumors [94], and activation of endogenous retrovirus LTRs, similarly to pluripotent cells, characterizes the HCC cell transcriptome [95].

Questions for the future The past decade has seen the rapid development of tools allowing us to rationally engineer mammalian www.sciencedirect.com

genomes; doing so wisely will be challenging. The examples above provide compelling evidence to reject the hypothesis that endogenous viral sequences are selfish DNA with no specific contribution to the reproductive success of its host organism [96]. There is thus a new set of practical questions facing this field, in addition to the need for new conceptual frameworks to better explain these observations [97,98]. Can we harness the existing systems controlling endogenous retroviruses to fight pathogenic exogenous ones [99]? One lead in this direction is the observation that genes endogenized from non-retroviral viruses are used to generate piRNA [70], a class of small RNA known to silence endogenous retroviruses and retrotransposons in mammals and, in insects, exogenous viruses [100]. Thus engineering animals with ‘artificially endogenized’ viral sequences in the proper genomic context may lead to heritable immunity. Modification of the porcine genome to remove endogenous retroviruses, a theorized impediment to xenotransplantation, has already been described [101]. Studying the organismlevel consequences of such modification, and in converse the implications of engineering hyperactive retroelements into the genome [102], will allow us to prospectively test ideas previously only amenable to teleological speculation. Similarly modified organisms may make ideal systems to test intriguing hypotheses about retroelement involvement in neural processes [103]. Additionally, studying mammals in which classes of retroelements have become extinct [104] or new types of retroelements have been added [105] may also provide insights (and help qualify overexuberance) about the physiological importance of retroelements. More proximally, more work to subdivide autoimmune disorders on the basis of genetic [106,107] and biochemical [108] evidence suggesting retroelement dysregulation is needed to allow therapeutic trials of reverse transcriptase inhibitors [59] and cGAS pathway inhibitors as these become available [60]. A better mechanistic understanding of the interplay between p53 mutation, retroelement transcription/reverse transcription, and interferon production may allow us to define cancer subtypes which may respond to available immunotherapeutics [109] or hold clues for new treatment approaches [110]. In summary, we propose that there is as much to learn about mammalian physiology from the forty percent of the genome contributed by reverse-transcribed RNA as we have from the two percent encoding protein.

Acknowledgements Space limitations prevented us from citing all of the recent advances in this field and we apologize to those whose work has been excluded. Research in Prof. Tomonaga’s laboratory is funded in part by Funding Program for Next Generation World-Leading Researchers (NEXT program), KAKENHI grant numbers 26253027 and 26670225, and the Core-to-Core Program A, Advanced Research Networks from the Japan Society for the Promotion of Science (JSPS); grants from Takeda Science Foundation. Current Opinion in Microbiology 2016, 31:176–183

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