Cumulative Roles for Epstein-Barr Virus, Human Endogenous Retroviruses, and Human Herpes Virus-6 in Driving an Inflammatory Cascade Underlying MS Pathogenesis.

Roles for viral infections and aberrant immune responses in driving localized neuroinflammation and neurodegeneration in multiple sclerosis (MS) are the focus of intense research. Epstein-Barr virus (EBV), as a persistent and frequently reactivating virus with major immunogenic influences and a near 100% epidemiological association with MS, is considered to play a leading role in MS pathogenesis, triggering localized inflammation near or within the central nervous system (CNS). This triggering may occur directly via viral products (RNA and protein) and/or indirectly via antigenic mimicry involving B-cells, T-cells and cytokine-activated astrocytes and microglia cells damaging the myelin sheath of neurons. The genetic MS-risk factor HLA-DR2b (DRB1*1501ß, DRA1*0101α) may contribute to aberrant EBV antigen-presentation and anti-EBV reactivity but also to mimicry-induced autoimmune responses characteristic of MS. A central role is proposed for inflammatory EBER1, EBV-miRNA and LMP1 containing exosomes secreted by viable reactivating EBV+ B-cells and repetitive release of EBNA1-DNA complexes from apoptotic EBV+ B-cells, forming reactive immune complexes with EBNA1-IgG and complement. This may be accompanied by cytokine- or EBV-induced expression of human endogenous retrovirus-W/-K (HERV-W/-K) elements and possibly by activation of human herpesvirus-6A (HHV-6A) in early-stage CNS lesions, each contributing to an inflammatory cascade causing the relapsing-remitting neuro-inflammatory and/or progressive features characteristic of MS. Elimination of EBV-carrying B-cells by antibody- and EBV-specific T-cell therapy may hold the promise of reducing EBV activity in the CNS, thereby limiting CNS inflammation, MS symptoms and possibly reversing disease. Other approaches targeting HHV-6 and HERV-W and limiting inflammatory kinase-signaling to treat MS are also being tested with promising results. This article presents an overview of the evidence that EBV, HHV-6, and HERV-W may have a pathogenic role in initiating and promoting MS and possible approaches to mitigate development of the disease.

Regulation of the expression of human endogenous retroviruses: elements in fetal development and a possible role in the development of cancer and neurological diseases.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral germline infections. Most HERV sequences are silenced in somatic cells, but interest is emerging on the involvement of HERV derived transcripts and proteins in human physiology and disease. A HERV-W encoded protein, syncytin-1, has been co-opted into fetal physiology, where it plays a role in trophoblast formation. Altered HERV transcription and expression of HERV derived proteins are associated with various cancer types and neurological diseases such as multiple sclerosis (MS). The implication of HERVs as potential mediators of both health and disease suggests important roles of regulatory mechanisms and alterations of these in physiological and pathological processes. The regulation of HERV sequences is mediated by a wide variety of mechanisms, and the focus of this review is on selected aspects of these, including epigenetic mechanisms such as CpG methylation and histone modifications of the HP1-H3K9me axis, viral transactivation events, and regulatory perspectives of transient stimuli in the microenvironment. Increasing knowledge of the regulation of HERV sequences will not only contribute to the understanding of complex pathogeneses, but also may pinpoint potential targets for better diagnosis and treatment in complex diseases as MS.

Origins and evolutionary consequences of ancient endogenous retroviruses.

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.

The HERV-K accessory protein Np9 controls viability and migration of teratocarcinoma cells.

Human endogenous retroviruses are remnants of ancient germline infections that make up approximately 8% of the modern human genome. The HERV-K (HML-2) family is one of the most recent entrants into the human germline, these viruses appear to be transcriptionally active, and HERV-K viral like particles (VLPs) are found in cell lines from a number of human malignancies. HERV-K VLPs were first found to be produced in teratocarcinoma cell lines, and since then teratocarcinoma has been thought of as the classical model for HERV-Ks, with the NCCIT teratocarcinoma cell line particularly known to produce VLPs. Treatment for teratocarcinoma has progressed since its discovery, with improved prognosis for patients. Since the introduction of platinum based therapy, first year survival has greatly improved even with disseminated disease; however, it is estimated that 20% to 30% of patients present with metastatic germ cell tumor relapse following initial treatments. Also, the toxicity associated with the use of chemotherapeutic agents used to treat germ cell tumors is still a major concern. In this study, we show that the depletion of the HERV-K accessory protein Np9 increases the sensitivity of NCCIT teratocarcinoma cells to bleomycin and cisplatin. While decreasing the expression of Np9 had only a modest effect on the baseline viability of the cells, the reduced expression of Np9 increased the sensitivity of the teratocarcinoma cells to environmental (serum starvation) and chemical (chemotherapeutic) stresses. Np9 is also essential to the migration of NCCIT teratocarcinoma cells: in a wound closure assay, reduced expression of Np9 resulted in cells migrating into the wound at a slower rate, whereas reintroduction of Np9 resulted in NCCIT cells migrating back into the wound in a manner similar to the control. These findings support the implication that the HERV-K accessory protein Np9 has oncogenic potential.

Amyloid Precursor Protein Overexpression in Down Syndrome Trophoblast Reduces Cell Invasiveness and Interferes with Syncytialization.

The placentas of Down syndrome (DS) pregnancies exhibit morphologic and functional abnormalities. Although the increase in dosage of certain genes on chromosome 21 has been associated with the DS phenotype, the effects on placenta have seldom been studied. Herein, we examine the expression of four dosage-sensitive genes (APP, ETS2, SOD1, and HMGN1) in normal and DS placentas. We demonstrated significant overexpression of amyloid precursor protein (APP) in DS placentas at RNA and protein levels by real-time quantitative PCR, Western blot analysis, and immunohistochemistry. Inducible APP overexpression trophoblast cell line models were established using a Tet-On system. APP induction in HTR-8/SVneo dose-dependently decelerated cell growth, enhanced apoptosis, and reduced cell migration and invasion when compared with the uninduced controls. Concomitantly, decreased ß-human chorionic gonadotropin in the culture medium was also detected on induction. Moreover, although forskolin treatment induced α/ß-human chorionic gonadotropin and syncytin expression in BeWo cells, such induction of syncytialization was inhibited by APP overexpression. E-cadherin immunofluorescence also demonstrated a decrease in syncytia formation in forskolin-treated BeWo-overexpressing APP. By liquid chromatography-tandem mass spectrometry, proteins related to cell-cell adhesion, protein translation, processing, and folding were found to be up-regulated in APP-induced HTR-8/SVneo clones. Our data demonstrated, for the first time, the effects of increased APP expression in DS placenta.

Cell-surface phosphatidylserine regulates osteoclast precursor fusion.

Bone-resorbing multinucleated osteoclasts that play a central role in the maintenance and repair of our bones are formed from bone marrow myeloid progenitor cells by a complex differentiation process that culminates in fusion of mononuclear osteoclast precursors. In this study, we uncoupled the cell fusion step from both pre-fusion stages of osteoclastogenic differentiation and the post-fusion expansion of the nascent fusion connections. We accumulated ready-to-fuse cells in the presence of the fusion inhibitor lysophosphatidylcholine and then removed the inhibitor to study synchronized cell fusion. We found that osteoclast fusion required the dendrocyte-expressed seven transmembrane protein (DC-STAMP)-dependent non-apoptotic exposure of phosphatidylserine at the surface of fusion-committed cells. Fusion also depended on extracellular annexins, phosphatidylserine-binding proteins, which, along with annexin-binding protein S100A4, regulated fusogenic activity of syncytin 1. Thus, in contrast to fusion processes mediated by a single protein, such as epithelial cell fusion in Caenorhabditis elegans, the cell fusion step in osteoclastogenesis is controlled by phosphatidylserine-regulated activity of several proteins.

Human endogenous retroviruses in the aetiology of MS.

Several lines of investigation have provided strong indications for an association between the immune-mediated, neurologic disease multiple sclerosis (MS) and human endogenous retroviruses (HERVs). Whether the relationship is causal is yet to be established. Endogenous retroviruses are pathogenic-in other species than the human. Several aspects of the activation and involvement of specific HERV families (HERV-H/F and HERV-W/MSRV) have been documented, both for cells in the periphery and in the central nervous system. Specific HERV-encoded genes and certain gene products (envelope proteins, Envs) appear strongly associated with the disease and have pathogenic potential. Most HERV sequences are non-functional, whereas some HERV loci have coding potential but remain quiescent in non-pathological conditions, so the importance of regulatory pathways and epigenetics involved in regulating HERV activation, de-repression, and also involvement of retroviral restriction factors, is emerging. Disease intervention by means of antiretrovirals has potential as a novel therapeutic strategy in MS treatment; this is compounded by the apparently reduced risk of MS in HIV infection as a consequence of therapy. Extensive studies of HERVs, their role in neurologic diseases, and their potential as therapeutic targets are needed.

Gamma-interferon-inducible, lysosome/endosome-localized thiolreductase, GILT, has anti-retroviral activity and its expression is counteracted by HIV-1.

The mechanism by which type II interferon (IFN) inhibits virus replications remains to be identified. Murine leukemia virus (MLV) replication was significantly restricted by γ-IFN, but not human immunodeficiency virus type 1 (HIV-1) replication. Because MLV enters host cells via endosomes, we speculated that certain cellular factors among γ-IFN-induced, endosome-localized proteins inhibit MLV replication. We found that γ-IFN-inducible lysosomal thiolreductase (GILT) significantly restricts HIV-1 replication as well as MLV replication by its thiolreductase activity. GILT silencing enhanced replication-defective HIV-1 vector infection and virion production in γ-IFN-treated cells, although γ-IFN did not inhibit HIV-1 replication. This result showed that GILT is required for the anti-viral activity of γ-IFN. Interestingly, GILT protein level was increased by γ-IFN in uninfected cells and env-deleted HIV-1-infected cells, but not in full-length HIV-1-infected cells. γ-IFN-induced transcription from the γ-IFN-activation sequence was attenuated by the HIV-1 Env protein. These results suggested that the γ-IFN cannot restrict HIV-1 replication due to the inhibition of γ-IFN signaling by HIV-1 Env. Finally, we found that 4,4'-dithiodipyridine (4-PDS), which inhibits S-S bond formation at acidic pH, significantly suppresses HIV-1 vector infection and virion production, like GILT. In conclusion, this study showed that GILT functions as a host restriction factor against the retroviruses, and a GILT mimic, 4-PDS, is the leading compound for the development of novel concept of anti-viral agents.

Expression and function of endogenous retroviruses in the placenta.

Although the expression of endogenous retroviruses in the placenta of numerous species was observed a long time ago, their physiological function during gestation was demonstrated only very recently. Expression of retroviral envelope proteins, also called syncytins, in the placenta allows generation of the multinuclear syncytiotrophoblast as an outer cellular layer of the placenta by fusion of the trophoblast cells. This fusion process is crucial for the development of the placenta and for successful pregnancy. It is still unclear whether the immunosuppressive properties of the transmembrane envelope protein of the endogenous retroviruses expressed in the placenta contribute to immunosuppression to prevent the rejection of the semiallotransplant embryo. The presence of placenta cells expressing retroviral envelope proteins surrounded by immune cells deep in the maternal tissue supports an immunosuppressive function. It is important to emphasize that during evolution different species utilized ('enslaved') different endogenous retroviruses and that two or more endogenous retroviruses are involved in placentogenesis in each species.

HERV-K(HML-2), a seemingly silent subtenant - but still waters run deep.

A large proportion of the human genome consists of endogenous retroviruses, some of which are well preserved, showing transcriptional activity, and expressing retroviral proteins. The HERV-K(HML-2) family represents the most intact members of these elements, with some having open and intact reading frames for viral proteins and the ability to form virus-like particles. Although generally suppressed in most healthy tissues by a variety of epigenetic processes and antiviral mechanisms, there is evidence that some members of this family are (at least partly) still active - particularly in certain stem cells and various tumors. This raises the possibility of their involvement in tumor induction or in developmental processes. In recent years, many new insights into this fascinating field have been attained, and this review focuses on new discoveries about coevolutionary events and intracellular defense mechanisms against HERV-K(HML-2) activity. We also describe what might occur when these mechanisms fail or become modulated by viral proteins or other viruses and discuss the new vistas opened up by the reconstitution of ancestral viral proteins and even complete HML-2 viruses.

Induction of ovine trophoblast cell fusion by fematrin-1 in vitro.

Endogenous retroviruses present in the genomes take a specific role in placental formation in various vertebrates, including bovine and sheep. Fematrin-1, which is the envelope (Env) protein of bovine endogenous retrovirus found in bovine placenta, is involved in the formation of fetomaternal hybrid cells in cattle placenta. This study was conducted to clarify whether fematrin-1 possesses fusogenic activity in trophoblast cells. Another question is whether Env proteins only have species-specific activity or not. For this, fematrin-1 gene was transfected in ovine trophoblast cells, and we examined fusogenic activity with Cos-7 cells. Although fematrin-1 fusogenic activity was detected in both neutral and acidic pH conditions, acidic condition significantly enhanced it. These activities were rather weaker than those of vesicular stomatitis virus G protein as a positive control. However, the ratio of fematrin-1 and vesicular stomatitis virus G protein fusion index was confirmed similar to those in the previous reports. Some fusion cells showed multinucleate cells. These results imply that fematrin-1 is involved in the formation of trophoblast hybrid cells even in different species trophoblastic cells.

Baton pass hypothesis: successive incorporation of unconserved endogenous retroviral genes for placentation during mammalian evolution.

It is well accepted that numerous RNAs derived from endogenous retroviruses (ERVs) are expressed in mammalian reproductive structures, particularly in the uterus, trophoblast, and placenta. Syncytin 1 and syncytin 2 in humans and syncytin A and syncytin B in mice are membrane proteins originating from Env genes of ERVs. These ERVs are involved in the fusion of trophoblast cells, resulting in multinucleated syncytiotrophoblast formation. Evidence accumulated indicates that syncytin-like fusogenic proteins are expressed in the placenta of rabbits, dogs/cats, ruminant ungulates, tenrecs, and opossums. The syncytin genes so far characterized are known to be endogenized to the host genome only within the past 12-80 million years, more recently than the appearance of mammalian placentas, estimated to be 160-180 million years ago. We speculate that ERVs including syncytin-like gene variants integrated into mammalian genomes in a locus-specific manner have replaced the genes previously responsible for cell fusion. We therefore propose the 'baton pass' hypothesis, in which multiple successive ERV variants 'take over' cell-fusion roles, resulting in increased trophoblast cell fusion, morphological variations in placental structures, and enhanced reproductive success in placental mammals.

Np9, a cellular protein of retroviral ancestry restricted to human, chimpanzee and gorilla, binds and regulates ubiquitin ligase MDM2.

Humans and primates are long-lived animals with long reproductive phases. One factor that appears to contribute to longevity and fertility in humans, as well as to cancer-free survival, is the transcription factor and tumor suppressor p53, controlled by its main negative regulator MDM2. However, p53 and MDM2 homologs are found throughout the metazoan kingdom from Trichoplacidae to Hominidae. Therefore the question arises, if p53/MDM2 contributes to the shaping of primate features, then through which mechanisms. Previous findings have indicated that the appearances of novel p53-regulated genes and wild-type p53 variants during primate evolution are important in this context. Here, we report on another mechanism of potential relevance. Human endogenous retrovirus K subgroup HML-2 (HERV-K(HML-2)) type 1 proviral sequences were formed in the genomes of the predecessors of contemporary Hominoidea and can be identified in the genomes of Nomascus leucogenys (gibbon) up to Homo sapiens. We previously reported on an alternative splicing event in HERV-K(HML-2) type 1 proviruses that can give rise to nuclear protein of 9 kDa (Np9). We document here the evolution of Np9-coding capacity in human, chimpanzee and gorilla, and show that the C-terminal half of Np9 binds directly to MDM2, through a domain of MDM2 that is known to be contacted by various cellular proteins in response to stress. Np9 can inhibit the MDM2 ubiquitin ligase activity toward p53 in the cell nucleus, and can support the transactivation of genes by p53. Our findings point to the possibility that endogenous retrovirus protein Np9 contributes to the regulation of the p53-MDM2 pathway specifically in humans, chimpanzees and gorillas.

Virus and cell fusion mechanisms.

In biomembrane fusion pathways, membranes are destabilized through insertions of amphipathic protein segments, lipid reorganization via hemifusion, protein restructuring, and dimpling of the membranes. Four classes of membrane proteins are known in virus and cell fusion. Class I virus-cell fusion proteins (fusogens) are α-helix-rich prefusion trimers that form coiled-coil structures that insert hydrophobic fusion peptides or loops (FPs or FLs) into membranes and refold into postfusion trimers. Class II virus-cell fusogens are ß-sheet-rich prefusion homo- or heterodimers that insert FLs into membranes, ending in postfusion trimers. Class III virus-cell fusogens are trimers with both α-helices and ß-sheets that dissociate into monomers, insert FLs into membranes, and oligomerize into postfusion trimers. Class IV reoviral cell-cell fusogens are small proteins with FLs that oligomerize to fuse membranes. Class I cell-cell fusogens (Syncytins) were captured by mammals from retroviruses, and class II cell-cell fusogens (EFF-1/AFF-1) fuse membranes via homotypic zippering. Mechanisms and fusogens for most cell fusion events are unknown.

Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia.

Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women.

Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation.

The development of the emerging field of 'paleovirology' allows biologists to reconstruct the evolutionary history of fossil endogenous retroviral sequences integrated within the genome of living organisms and has led to the retrieval of conserved, ancient retroviral genes 'exapted' by ancestral hosts to fulfil essential physiological roles, syncytin genes being undoubtedly among the most remarkable examples of such a phenomenon. Indeed, syncytins are 'new' genes encoding proteins derived from the envelope protein of endogenous retroviral elements that have been captured and domesticated on multiple occasions and independently in diverse mammalian species, through a process of convergent evolution. Knockout of syncytin genes in mice provided evidence for their absolute requirement for placenta development and embryo survival, via formation by cell-cell fusion of syncytial cell layers at the fetal-maternal interface. These genes of exogenous origin, acquired 'by chance' and yet still 'necessary' to carry out a basic function in placental mammals, may have been pivotal in the emergence of mammalian ancestors with a placenta from egg-laying animals via the capture of a founding retroviral env gene, subsequently replaced in the diverse mammalian lineages by new env-derived syncytin genes, each providing its host with a positive selective advantage.

Refrex-1, a soluble restriction factor against feline endogenous and exogenous retroviruses.

The host defense against viral infection is acquired during the coevolution or symbiosis of the host and pathogen. Several cellular factors that restrict retroviral infection have been identified in the hosts. Feline leukemia virus (FeLV) is a gammaretrovirus that is classified into several receptor interference groups, including a novel FeLV-subgroup D (FeLV-D) that we recently identified. FeLV-D is generated by transduction of the env gene of feline endogenous gammaretrovirus of the domestic cat (ERV-DCs) into FeLV. Some ERV-DCs are replication competent viruses which are present and hereditary in cats. We report here the determination of new viral receptor interference groups and the discovery of a soluble antiretroviral factor, termed Refrex-1. Detailed analysis of FeLV-D strains and ERV-DCs showed two receptor interference groups that are distinct from other FeLV subgroups, and Refrex-1 specifically inhibited one of them. Refrex-1 is characterized as a truncated envelope protein of ERV-DC and includes the N-terminal region of surface unit, which is a putative receptor-binding domain, but lacks the transmembrane region. Refrex-1 is efficiently secreted from the cells and appears to cause receptor interference extracellularly. Two variants of Refrex-1 encoded by provirus loci, ERV-DC7 and DC16, are expressed in a broad range of feline tissues. The host retains Refrex-1 as an antiretroviral factor, which may potentially prevent reemergence of the ERVs and the emergence of novel ERV-related viruses in cats. Refrex-1 may have been acquired during endogenization of ERV-DCs and may play an important role in retroviral restriction and antiviral defense in cats.

Differential evolutionary fate of an ancestral primate endogenous retrovirus envelope gene, the EnvV syncytin, captured for a function in placentation.

Syncytins are envelope genes of retroviral origin that have been co-opted for a role in placentation. They promote cell-cell fusion and are involved in the formation of a syncytium layer--the syncytiotrophoblast--at the materno-fetal interface. They were captured independently in eutherian mammals, and knockout mice demonstrated that they are absolutely required for placenta formation and embryo survival. Here we provide evidence that these "necessary" genes acquired "by chance" have a definite lifetime with diverse fates depending on the animal lineage, being both gained and lost in the course of evolution. Analysis of a retroviral envelope gene, the envV gene, present in primate genomes and belonging to the endogenous retrovirus type V (ERV-V) provirus, shows that this captured gene, which entered the primate lineage >45 million years ago, behaves as a syncytin in Old World monkeys, but lost its canonical fusogenic activity in other primate lineages, including humans. In the Old World monkeys, we show--by in situ analyses and ex vivo assays--that envV is both specifically expressed at the level of the placental syncytiotrophoblast and fusogenic, and that it further displays signs of purifying selection based on analysis of non-synonymous to synonymous substitution rates. We further show that purifying selection still operates in the primate lineages where the gene is no longer fusogenic, indicating that degeneracy of this ancestral syncytin is a slow, lineage-dependent, and multi-step process, in which the fusogenic activity would be the first canonical property of this retroviral envelope gene to be lost.

From ancestral infectious retroviruses to bona fide cellular genes: role of the captured syncytins in placentation.

During their replication, infectious retroviruses insert a reverse-transcribed cDNA copy of their genome, a "provirus", into the genome of their host. If the infected cell belongs to the germline, the integrated provirus can become "fixed" within the host genome as an endogenous retrovirus and be transmitted vertically to the progeny in a Mendelian fashion. Based on the numerous proviral sequences that are recovered within the genomic DNA of vertebrates--up to ten percent in the case of mammals--such events must have occurred repeatedly during the course of millions of years of evolution. Although most of the ancient proviral sequences have been disrupted, a few "endogenized" retroviral genes are conserved and still encode functional proteins. In this review, we focus on the recent discovery of genes derived from the envelope glycoprotein-encoding (env) genes of endogenous retroviruses that have been domesticated by mammals to carry out an essential function in placental development. They were called syncytins based on the membrane fusogenic capacity that they have kept from their parental env gene and which contributes to the formation of the placental fused cell layer called the syncytiotrophoblast, at the materno-fetal interface. Remarkably, the capture of syncytin or syncytin-like genes, sometimes as pairs, was found to have occurred independently from different endogenous retroviruses in diverse mammalian lineages such as primates--including humans--, muroids, leporids, carnivores, caviids, and ovis, between around 10 and 85 million years ago. Knocking out one or both mouse syncytin-A and -B genes provided evidence that they indeed play a critical role in placentation. We discuss the possibility that the immunosuppressive domain embedded within retroviral envelope glycoproteins and conserved in syncytin proteins, may be involved in the tolerance of the fetus by the maternal immune system. Finally, we speculate that the capture of a founding syncytin-like gene could have been instrumental in the dramatic transition from egg-laying to placental mammals.

Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora.

Syncytins are envelope protein genes of retroviral origin that have been captured for a function in placentation. Two such genes have already been identified in simians, two distinct, unrelated genes have been identified in Muridae, and a fifth gene has been identified in the rabbit. Here, we searched for similar genes in the Laurasiatheria clade, which diverged from Euarchontoglires--primates, rodents, and lagomorphs--shortly after mammalian radiation (100 Mya). In silico search for envelope protein genes with full-coding capacity within the dog and cat genomes identified several candidate genes, with one common to both species that displayed placenta-specific expression, which was revealed by RT-PCR analysis of a large panel of tissues. This gene belongs to a degenerate endogenous retroviral element, with precise proviral integration at a site common to dog and cat. Cloning of the gene for an ex vivo pseudotype assay showed fusogenicity on both dog and cat cells. In situ hybridization on placenta sections from both species showed specific expression at the level of the invasive fetal villi within the placental junctional zone, where trophoblast cells fuse into a syncytiotrophoblast layer to form the maternofetal interface. Finally, we show that the gene is conserved among a series of 26 Carnivora representatives, with evidence for purifying selection and conservation of fusogenic activity. The gene is not found in the Pholidota order and, therefore, it was captured before Carnivora radiation, between 60 and 85 Mya. This gene is the oldest syncytin gene identified to date, and it is the first in a new major clade of eutherian mammals.