Cell-Int: a cell-cell interaction assay to identify native membrane protein interactions.

Intercellular protein-protein interactions (PPIs) have pivotal roles in biological functions and diseases. Membrane proteins are therefore a major class of drug targets. However, studying such intercellular PPIs is challenging because of the properties of membrane proteins. Current methods commonly use purified or modified proteins that are not physiologically relevant and hence might mischaracterize interactions occurring in vivo. Here, we describe Cell-Int: a cell interaction assay for studying plasma membrane PPIs. The interaction signal is measured through conjugate formation between two populations of cells each expressing either a ligand or a receptor. In these settings, membrane proteins are in their native environment thus being physiologically relevant. Cell-Int has been applied to the study of diverse protein partners, and enables to investigate the inhibitory potential of blocking antibodies, as well as the retargeting of fusion proteins for therapeutic development. The assay was also validated for screening applications and could serve as a platform for identifying new protein interactors.

Sodium-dependent phosphate transporter PiT1/SLC20A1 as the receptor for the endogenous retroviral envelope syncytin-B involved in mouse placenta formation.

Syncytins are envelope genes of retroviral origin that play a critical role in the formation of a syncytial structure at the fetomaternal interface via their fusogenic activity. The mouse placenta is unique among placental mammals since the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one observed in all other hemochorial placentae. Each layer specifically expresses a distinct mouse syncytin, namely syncytin-A (SynA) for ST-I and syncytin-B (SynB) for ST-II, which have been shown to be essential to placentogenesis and embryonic development. The cellular receptor for SynA has been identified as the membrane protein LY6E and is not the receptor for SynB. Here, by combining a cell-cell fusion assay with the screening of a human ORFeome-derived expression library, we identified the transmembrane multipass sodium-dependent phosphate transporter 1 PiT1/SLC20A1 as the receptor for SynB. Transfection of cells with the cloned receptor, but not the closely related PiT2/SLC20A2, leads to their fusion with cells expressing SynB, with no cross-reactive fusion activity with SynA. The interaction between the two partners was further demonstrated by immunoprecipitation. PiT1/PiT2 chimera and truncation experiments identified the PiT1 N-terminus as the major determinant for SynB-mediated fusion. RT-qPCR analysis of PiT1 expression on a panel of mouse adult and fetal tissues revealed a concomitant increase of PiT1 and SynB specifically in the developing placenta. Finally, electron microscopy analysis of the placenta of PiT1 null embryo before they die (E11.5) disclosed default of ST-II formation with lack of syncytialization, as previously observed in cognate SynB null placenta, and consistent with the present identification of PiT1 as the SynB partner.IMPORTANCESyncytins are envelope genes of endogenous retroviruses, coopted for a physiological function in placentation. They are fusogenic proteins that mediate cell-cell fusion by interacting with receptors present on the partner cells. Here, by devising an in vitro fusion assay that enables the screening of an ORFeome-derived expression library, we identified the long-sought receptor for syncytin-B (SynB), a mouse syncytin responsible for syncytiotrophoblast formation at the fetomaternal interface of the mouse placenta. This protein - PiT1/SLC20A1 - is a multipass transmembrane protein, also known as the receptor for a series of infectious retroviruses. Its profile of expression is consistent with a role in both ancestral endogenization of a SynB founder retrovirus and present-day mouse placenta formation, with evidence-in PiT1 knockout mice-of unfused cells at the level of the cognate placental syncytiotrophoblast layer.

IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases.

Interferon-induced transmembrane proteins (IFITMs) are restriction factors that block many viruses from entering cells. High levels of type I interferon (IFN) are associated with adverse pregnancy outcomes, and IFITMs have been shown to impair the formation of syncytiotrophoblast. Here, we examine whether IFITMs affect another critical step of placental development, extravillous cytotrophoblast (EVCT) invasion. We conducted experiments using in vitro/ex vivo models of EVCT, mice treated in vivo with the IFN-inducer poly (I:C), and human pathological placental sections. Cells treated with IFN-ß demonstrated upregulation of IFITMs and reduced invasive abilities. Transduction experiments confirmed that IFITM1 contributed to the decreased cell invasion. Similarly, migration of trophoblast giant cells, the mouse equivalent of human EVCTs, was significantly reduced in poly (I:C)-treated mice. Finally, analysis of CMV- and bacterial-infected human placentas revealed upregulated IFITM1 expression. These data demonstrate that high levels of IFITM1 impair trophoblast invasion and could explain the placental dysfunctions associated with IFN-mediated disorders.

Performance assessment and validation of a plaque reduction neutralization test (PRNT) in support to yellow fever diagnostic and vaccine clinical trials.

Yellow fever (YF) virus is a mosquito-borne virus belonging to the Flaviviridae family that circulates in tropical and subtropical areas of Africa and South America. Despite the availability of an effective vaccine, YF remains a threat to travelers, residents of endemic areas, and unvaccinated populations. YF vaccination and natural infection both induce the production of neutralizing antibodies. Serological diagnostic methods detecting YF virus-specific antibodies demonstrate high levels of cross-reactivities with other flaviviruses. To date, the plaque reduction neutralization test (PRNT) is the most specific serological test for the differentiation of flavivirus infections and is considered the reference method for detecting YF neutralizing antibodies and assessing the protective immune response following vaccination. In this study, we developed and validated a YF PRNT. We optimized different parameters including cell concentration and virus-serum neutralization time period and then assessed the intra- and inter-assay precisions, dilutability, specificity, and lower limit of quantification (LLOQ) using international standard YF serum, sera from vaccinees and human specimens collected through YF surveillance. The YF PRNT has shown good robustness and 100% of intra-assay precision, 95.6% of inter-assay precision, 100% of specificity, 100% of LLOQ, and 95.3% of dilutability. The test is, therefore, suitable for use in the YF diagnostic as well as evaluation of the YF vaccine neutralizing antibody response and risk assessment studies.

IFITM proteins inhibit placental syncytiotrophoblast formation and promote fetal demise.

Elevated levels of type I interferon (IFN) during pregnancy are associated with intrauterine growth retardation, preterm birth, and fetal demise through mechanisms that are not well understood. A critical step of placental development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast (ST) layer. Fusion is mediated by syncytins, proteins deriving from ancestral endogenous retroviral envelopes. Using cultures of human trophoblasts or mouse cells, we show that IFN-induced transmembrane proteins (IFITMs), a family of restriction factors blocking the entry step of many viruses, impair ST formation and inhibit syncytin-mediated fusion. Moreover, the IFN inducer polyinosinic:polycytidylic acid promotes fetal resorption and placental abnormalities in wild-type but not in Ifitm-deleted mice. Thus, excessive levels of IFITMs may mediate the pregnancy complications observed during congenital infections and other IFN-induced pathologies.

Role of the captured retroviral envelope syncytin-B gene in the fusion of osteoclast and giant cell precursors and in bone resorption, analyzed ex vivo and in vivo in syncytin-B knockout mice.

Syncytin-A and -B are envelope genes of retroviral origin that have been captured in evolution for a role in placentation. They trigger cell-cell fusion and were shown to be essential for the formation of the syncytiotrophoblast layer during mouse placenta formation. Syncytin-A and -B expression has been described in other tissues and their highly fusogenic properties suggested that they might be involved in the fusion of other cell types. Here, taking advantage of mice knocked out for syncytin-B, SynB-/- mice, we investigated the potential role of syncytin-B in the fusion of cells from the monocyte/macrophage lineage into multinucleated osteoclasts (OCs) -in bone- or multinucleated giant cells -in soft tissues. In ex vivo experiments, a significant reduction in fusion index and in the number of multinucleated OCs and giant cells was observed as soon as Day3 in SynB-/- as compared to wild-type cell cultures. Interestingly, the number of nuclei per multinucleated OC or giant cell remained unchanged. These results, together with the demonstration that syncytin-B expression is maximal in the first 2 days of OC differentiation, argue for syncytin-B playing a role in the fusion of OC and giant cell mononucleated precursors, at initial stages. Finally, ex vivo, the observed reduction in multinucleated OC number had no impact on the expression of OC differentiation markers, and a dentin resorption assay did not evidence any difference in the osteoclastic resorption activity, suggesting that syncytin-B is not required for OC activity. In vivo, syncytin-B was found to be expressed in the periosteum of embryos at embryonic day 16.5, where TRAP-positive cells were observed. Yet, in adults, no significant reduction in OC number or alteration in bone phenotype was observed in SynB-/- mice. In addition, SynB-/- mice did not show any change in the number of foreign body giant cells (FBGCs) that formed in response to implantation of foreign material, as compared to wild-type mice. Altogether the results suggest that in addition to its essential role in placenta formation, syncytin-B plays a role in OCs and macrophage fusion; yet it is not essential in vivo for OC and FBGC formation, or maintenance of bone homeostasis, at least under the conditions tested.

Capture of a Hyena-Specific Retroviral Envelope Gene with Placental Expression Associated in Evolution with the Unique Emergence among Carnivorans of Hemochorial Placentation in Hyaenidae.

Capture of retroviral envelope genes from endogenous retroviruses has played a role in the evolution of mammals, with evidence for the involvement of these genes in the formation of the maternofetal interface of the placenta. It has been shown that the diversity of captured genes is likely to be responsible for the diversity of placental structures, ranging from poorly invasive (epitheliochorial) to highly invasive (hemochorial), with an intermediate state (endotheliochorial) as found in carnivorans. The latter recapitulate part of this evolution, with the hyena being the sole carnivoran with a hemochorial placenta. In this study, we performed RNA sequencing on hyena placental transcripts and searched for endogenous retroviral envelope genes that have been captured specifically in the Hyaenidae clade and are not found in any other carnivoran. We identified an envelope gene that is expressed in the placenta at the level of the maternofetal interface, as evidenced by in situ hybridization/immunohistochemistry. The gene entry is coincidental with the emergence of the Hyaenidae clade 30 million years ago (Mya), being found at the same genomic locus in all 4 extant hyena species. Its coding sequence has further been maintained during all of Hyaenidae evolution. It is not found in any of the 30 other carnivorans-both Felidae and Canidae-that we screened. This envelope protein does not disclose any fusogenic activity in ex vivo assays, at variance with the syncytin-Car1 gene, which is found in all carnivorans, including the hyena, in which it is still present, transcriptionally active in the placenta, and fusogenic. Together, the present results illustrate the permanent renewal of placenta-specific genes by retroviral capture and de facto provide a candidate gene for the endotheliochorial to hemochorial transition of Hyaenidae among carnivorans.IMPORTANCE The placenta is the most diverse organ among mammals, due in part to stochastic capture of retroviral envelope genes. In carnivorans, capture of syncytin-Car1 took place 80 Mya. It is fusogenic, expressed at the syncytialized placental maternofetal interface, and conserved among all carnivorans, consistent with their shared endotheliochorial placenta. Hyenas are a remarkable exception, with a highly invasive hemochorial placenta, as found in humans, where disruption of maternal blood vessels results in maternal blood bathing the syncytial maternofetal interface. In this study, we identified a retroviral envelope gene capture and exaptation that took place about 30 Mya and is coincident with the emergence of the Hyaenidae, being conserved in all extant hyena species. It is expressed at the maternofetal interface in addition to the shared syncytin-Car1 gene. This new env gene, not present in any other carnivoran, is a likely candidate to be responsible for the specific structure of the hyena placenta.

An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard.

Syncytins are envelope genes from endogenous retroviruses that have been captured during evolution for a function in placentation. They have been found in all placental mammals in which they have been searched, including marsupials. Placental structures are not restricted to mammals but also emerged in some other vertebrates, most frequently in lizards, such as the viviparous Mabuya Scincidae. Here, we performed high-throughput RNA sequencing of a Mabuya placenta transcriptome and screened for the presence of retroviral env genes with a full-length ORF. We identified one such gene, which we named "syncytin-Mab1," that has all the characteristics expected for a syncytin gene. It encodes a membrane-bound envelope protein with fusogenic activity ex vivo, is expressed at the placental level as revealed by in situ hybridization and immunohistochemistry, and is conserved in all Mabuya species tested, spanning over 25 My of evolution. Its cognate receptor, required for its fusogenic activity, was searched for by a screening assay using the GeneBridge4 human/Chinese hamster radiation hybrid panel and found to be the MPZL1 gene, previously identified in mammals as a signal-transducing transmembrane protein involved in cell migration. Together, these results show that syncytin capture is not restricted to placental mammals, but can also take place in the rare nonmammalian vertebrates in which a viviparous placentotrophic mode of reproduction emerged. It suggests that similar molecular tools have been used for the convergent evolution of placentation in independently evolved and highly distant vertebrates.

A Cell Fusion-Based Screening Method Identifies Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A.

Syncytin genes are envelope genes of retroviral origin that have been exapted for a role in placentation. They are involved in the formation of a syncytial structure (the syncytiotrophoblast) at the fetomaternal interface via their fusogenic activity. The mouse placenta is unique among placental mammals since the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one, as observed in humans and all other hemochorial placentae. Each layer specifically expresses a distinct mouse syncytin, namely, syncytin-A (SynA) for ST-I and syncytin-B (SynB) for ST-II, which have been shown to be essential to placentogenesis and embryo survival. Their cognate cellular receptors, which are necessary to mediate cell-cell fusion and syncytiotrophoblast formation, are still unknown. By devising a sensitive method that combines a cell-cell fusion assay with the screening of a mouse cDNA library, we succeeded in identifying the glycosylphosphatidylinositol (GPI)-anchored membrane protein lymphocyte antigen 6E (Ly6e) as a candidate receptor for SynA. Transfection of cells with the cloned receptor led to their fusion to cells expressing SynA, with no cross-reactive fusion activity with SynB. Knocking down Ly6e greatly reduced SynA-induced cell fusion, thus suggesting that Ly6e is the sole receptor for SynA in vivo Interaction of SynA with Ly6e was further demonstrated by a competition assay using the soluble ectodomain of Ly6e. Finally, reverse transcription-quantitative PCR (RT-qPCR) analysis of Ly6e expression on a representative panel of mouse tissues shows that it is significantly expressed in the mouse placenta together with SynA.IMPORTANCE Syncytin genes are envelope genes of endogenous retroviruses, co-opted for a physiological function in placentation. Syncytins are fusogenic proteins that mediate cell-cell fusion by interacting with receptors present on the partner cells. Here, by devising a sensitive in vitro fusion assay that enables the high-throughput screening of normalized cDNA libraries, we identified the long-sought receptor for syncytin-A (SynA), a mouse syncytin responsible for syncytiotrophoblast formation at the maternofetal interface of the mouse placenta. This protein, Ly6e (lymphocyte antigen 6E), is a GPI-anchored membrane protein, and small interfering RNA (siRNA) experiments targeting its deletion as well as a decoy assay using a recombinant soluble receptor show that Ly6e is the necessary and sufficient partner of SynA. Its profile of expression is consistent with a role in both ancestral endogenization of a SynA founder retrovirus and present-day placenta formation. This study provides a powerful general method to identify genes involved in cell-cell fusion processes.

Real-Time RT-PCR Assays for Detection and Genotyping of West Nile Virus Lineages Circulating in Africa.

West Nile virus (WNV) is an emerging arbovirus, circulating worldwide between birds and mosquitoes, which impacts human and animal health. Since the mid-1990s, WNV outbreaks have emerged in Europe and America and represent currently the primary cause of encephalitis in the United States. WNV exhibits a great genetic diversity with at least eight different lineages circulating in the world, and four (1, 2, Koutango, and putative new) are present in Africa. These different WNV lineages are not readily differentiated by serology, and thus, rapid molecular tools are required for diagnostic. We developed here real-time RT-PCR assays for detection and genotyping of African WNV lineages. The specificity of the assays was tested using other flaviviruses circulating in Africa. The sensitivity was determined by testing serial 10-fold dilutions of viruses and RNA standards. The assays provided good specificity and sensitivity and the analytical detection limit was 10 copies/reaction. The RT-PCR assays allowed the detection and genotyping of all WNV isolates in culture medium, human serum, and vertebrate tissues, as well as in field and experimental mosquito samples. Comparing the ratios of genome copy number/infectious virion (plaque-forming units), our study finally revealed new insight on the replication of these different WNV lineages in mosquito cells. Our RT-PCR assays are the first ones allowing the genotyping of all WNV African variants, and this may have important applications in surveillance and epidemiology in Africa and also for monitoring of their emergence in Europe and other continents.

Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice.

Syncytins are envelope genes from endogenous retroviruses, "captured" for a role in placentation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncytiotrophoblast) at the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucleated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20-40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals.

Genome-Wide Screening of Retroviral Envelope Genes in the Nine-Banded Armadillo (Dasypus novemcinctus, Xenarthra) Reveals an Unfixed Chimeric Endogenous Betaretrovirus Using the ASCT2 Receptor.

UNLABELLED: Retroviruses enter host cells through the interaction of their envelope (Env) protein with a cell surface receptor, which triggers the fusion of viral and cellular membranes. The sodium-dependent neutral amino acid transporter ASCT2 is the common receptor of the large RD114 retrovirus interference group, whose members display frequent env recombination events. Germ line retrovirus infections have led to numerous inherited endogenous retroviruses (ERVs) in vertebrate genomes, which provide useful insights into the coevolutionary history of retroviruses and their hosts. Rare ERV-derived genes display conserved viral functions, as illustrated by the fusogenic syncytin env genes involved in placentation. Here, we searched for functional env genes in the nine-banded armadillo (Dasypus novemcinctus) genome and identified dasy-env1.1, which clusters with RD114 interference group env genes and with two syncytin genes sharing ASCT2 receptor usage. Using ex vivo pseudotyping and cell-cell fusion assays, we demonstrated that the Dasy-Env1.1 protein is fusogenic and can use both human and armadillo ASCT2s as receptors. This gammaretroviral env gene belongs to a provirus with betaretrovirus-like features, suggesting acquisition through recombination. Provirus insertion was found in several Dasypus species, where it has not reached fixation, whereas related family members integrated before diversification of the genus Dasypus >12 million years ago (Mya). This newly described ERV lineage is potentially useful as a population genetic marker. Our results extend the usage of ASCT2 as a retrovirus receptor to the mammalian clade Xenarthra and suggest that the acquisition of an ASCT2-interacting env gene is a major selective force driving the emergence of numerous chimeric viruses in vertebrates. IMPORTANCE: Retroviral infection is initiated by the binding of the viral envelope glycoprotein to a host cell receptor(s), triggering membrane fusion. Ancient germ line infections have generated numerous endogenous retroviruses (ERVs) in nearly all vertebrate genomes. Here, we report a previously uncharacterized ERV lineage from the genome of a xenarthran species, the nine-banded armadillo (Dasypus novemcinctus). It entered the Dasypus genus >12 Mya, with one element being inserted more recently in some Dasypus species, where it could serve as a useful marker for population genetics. This element exhibits an env gene, acquired by recombination events, with conserved viral fusogenic properties through binding to ASCT2, a receptor used by a wide range of recombinant retroviruses infecting other vertebrate orders. This specifies the ASCT2 transporter as a successful receptor for ERV endogenization and suggests that ASCT2-binding env acquisition events have favored the emergence of numerous chimeric viruses in a wide range of species.

Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials.

Syncytins are genes of retroviral origin captured by eutherian mammals, with a role in placentation. Here we show that some marsupials-which are the closest living relatives to eutherian mammals, although they diverged from the latter ∼190 Mya-also possess a syncytin gene. The gene identified in the South American marsupial opossum and dubbed syncytin-Opo1 has all of the characteristic features of a bona fide syncytin gene: It is fusogenic in an ex vivo cell-cell fusion assay; it is specifically expressed in the short-lived placenta at the level of the syncytial feto-maternal interface; and it is conserved in a functional state in a series of Monodelphis species. We further identify a nonfusogenic retroviral envelope gene that has been conserved for >80 My of evolution among all marsupials (including the opossum and the Australian tammar wallaby), with evidence for purifying selection and conservation of a canonical immunosuppressive domain, but with only limited expression in the placenta. This unusual captured gene, together with a third class of envelope genes from recently endogenized retroviruses-displaying strong expression in the uterine glands where retroviral particles can be detected-plausibly correspond to the different evolutionary statuses of a captured retroviral envelope gene, with only syncytin-Opo1 being the present-day bona fide syncytin active in the opossum and related species. This study would accordingly recapitulate the natural history of syncytin exaptation and evolution in a single species, and definitely extends the presence of such genes to all major placental mammalian clades.

The captured retroviral envelope syncytin-A and syncytin-B genes are conserved in the Spalacidae together with hemotrichorial placentation.

Syncytins are fusogenic envelope (env) genes of retroviral origin that have been captured for a function in placentation. Multiple independent events of syncytin gene capture were found to have occurred in primates, rodents, lagomorphs, carnivores, and ruminants. In the mouse, two syncytin-A and -B genes are present, which trigger the formation of the two-layered placental syncytiotrophoblast at the maternal-fetal interface, a structure classified as hemotrichorial. Here, we identified syncytin-A and -B orthologous genes in the genome of all Muroidea species analyzed, thus dating their capture back to about at least 40 million years ago, with evidence that they evolved under strong purifying selection. We further show, in the divergent Spalacidae lineage (blind mole rats [Spalax]), that both syncytins have conserved placenta-specific expression, as revealed by RT-PCR analysis of a panel of Spalax galili tissues, and display fusogenic activity, using ex vivo cell-cell fusion assays. Refined analysis of the placental architecture and ultrastructure revealed that the Spalax placenta displays a hemotrichorial organization of the interhemal membranes, as similarly observed for other Muroidea species, yet with only one trophoblastic cell layer being clearly syncytialized. In situ hybridization experiments further localized syncytin transcripts at the level of these differentiated interhemal membranes. These findings argue for a role of syncytin gene capture in the establishment of the original hemotrichorial placenta of Muroidea, and more generally in the diversity of placental structures among mammals.

Retroviral envelope syncytin capture in an ancestrally diverged mammalian clade for placentation in the primitive Afrotherian tenrecs.

Syncytins are fusogenic envelope (env) genes of retroviral origin that have been captured for a function in placentation. Syncytins have been identified in Euarchontoglires (primates, rodents, Leporidae) and Laurasiatheria (Carnivora, ruminants) placental mammals. Here, we searched for similar genes in species that retained characteristic features of primitive mammals, namely the Malagasy and mainland African Tenrecidae. They belong to the superorder Afrotheria, an early lineage that diverged from Euarchotonglires and Laurasiatheria 100 Mya, during the Cretaceous terrestrial revolution. An in silico search for env genes with full coding capacity within a Tenrecidae genome identified several candidates, with one displaying placenta-specific expression as revealed by RT-PCR analysis of a large panel of Setifer setosus tissues. Cloning of this endogenous retroviral env gene demonstrated fusogenicity in an ex vivo cell-cell fusion assay on a panel of mammalian cells. Refined analysis of placental architecture and ultrastructure combined with in situ hybridization demonstrated specific expression of the gene in multinucleate cellular masses and layers at the materno-fetal interface, consistent with a role in syncytium formation. This gene, which we named "syncytin-Ten1," is conserved among Tenrecidae, with evidence of purifying selection and conservation of fusogenic activity. To our knowledge, it is the first syncytin identified to date within the ancestrally diverged Afrotheria superorder.

Capture of syncytin-Mar1, a fusogenic endogenous retroviral envelope gene involved in placentation in the Rodentia squirrel-related clade.

Syncytin genes are fusogenic envelope protein (env) genes of retroviral origin that have been captured for a function in placentation. Within rodents, two such genes have previously been identified in the mouse-related clade, allowing a demonstration of their essential role via knockout mice. Here, we searched for similar genes in a second major clade of the Rodentia order, the squirrel-related clade, taking advantage of the complete sequencing of the ground squirrel Ictidomys tridecemlineatus genome. In silico search for env genes with full coding capacity identified several candidate genes with one displaying placenta-specific expression, as revealed by quantitative reverse transcription-PCR analysis of a large panel of tissues. This gene belongs to a degenerate endogenous retroviral element, with recognizable hallmarks of an integrated provirus. Cloning of the gene in an expression vector for ex vivo cell-cell fusion and pseudotype assays demonstrated fusogenicity on a large panel of mammalian cells. In situ hybridization on placenta sections showed specific expression in domains where trophoblast cells fuse into a syncytiotrophoblast at the fetomaternal interface, consistent with a role in syncytium formation. Finally, we show that the gene is conserved among the tribe Marmotini, thus dating its capture back to about at least 25 million years ago, with evidence for purifying selection and conservation of fusogenic activity. This gene that we named syncytin-Mar1 is distinct from all seven Syncytin genes identified to date in eutherian mammals and is likely to be a major effector of placentation in its related clade. Importance: Syncytin genes are fusogenic envelope genes of retroviral origin, ancestrally captured for a function in placentation. Within rodents, two such genes had been previously identified in the mouse-related clade. Here, in the squirrel-related rodent clade, we identified the envelope gene of an endogenous retrovirus with all the features of a Syncytin: it is specifically expressed in the placenta of the woodchuck Marmota monax, at the level of cells fusing into a syncytium; it can trigger cell-cell and virus-cell fusion ex vivo; and it has been conserved for >25 million years of evolution, suggesting an essential role in its host physiology. Remarkably, syncytin-Mar1 is unrelated to all other Syncytin genes identified thus far in mammals (primates, muroids, carnivores, and ruminants). These results extend the range of retroviral envelope gene "domestication" in mammals and show that these events occurred independently, on multiple occasions during evolution to improve placental development in a process of convergent evolution.

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.

Comparative full length genome sequence analysis of Usutu virus isolates from Africa.

BACKGROUND: Usutu virus (USUV), a flavivirus belonging to the Japanese encephalitis serocomplex, was identified in South Africa in 1959 and reported for the first time in Europe in 2001. To date, full length genome sequences have been available only for the reference strain from South Africa and a single isolate from each of Austria, Hungary, and Italy. METHODS: We sequenced four USUV isolates from Senegal and the Central African Republic (CAR) between 1974 and 2007 and compared the sequence data to USUV strains from Austria, Hungary, Italy, and South Africa using a Bayesian Markov chain Monte Carlo method. We further clarified the taxonomic status of a USUV strain isolated in CAR in 1969 and proposed earlier as a subtype of USUV due to an asymetric serological cross-reactivity with USUV reference strain. RESULTS: A comparison of the four newly obtained USUV sequences with those from SouthAfrica_1959, Vienna_2001, Budapest_2005, and Italy_2009 revealed that they are all 96-99% and 99% similar at the nucleotide and amino acid levels, respectively. The phylogenetic relationships between these sequences indicated that a strain isolated in Senegal in 1993 is most closely related to the USUV strains detected in Europe. Analysis of a strain isolated from a human in CAR in 1981 (CAR_1981) revealed the presence of specific amino acid substitutions and a deletion in the 3' noncoding region. This is the first fully sequenced human USUV isolate.The putative USUV subtype, CAR_1969, was 81% and 94% identical at the nucleotide and amino acid levels, respectively, compared to the other USUV strains. Our phylogenetic analyses support the serological identification of CAR_1969 as a subtype of USUV. CONCLUSIONS: In this study, we investigate the genetic diversity of USUV in Africa and the phylogenetic relationship of isolates from Africa and Europe for the first time. The results suggest a low genetic diversity within USUV, the existence of a distinct USUV subtype strain, and support the hypothesis that USUV was introduced to Europe from Africa. Further sequencing and analysis of USUV isolates from other African countries would contribute to a better understanding of its genetic diversity and geographic distribution.

Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants.

Syncytins are envelope genes of retroviral origin that have been co-opted for a role in placentation and likely contribute to the remarkable diversity of placental structures. Independent capture events have been identified in primates, rodents, lagomorphs, and carnivores, where they are involved in the formation of a syncytium layer at the fetomaternal interface via trophoblast cell-cell fusion. We searched for similar genes within the suborder Ruminantia where the placenta lacks an extended syncytium layer but displays a heterologous cell-fusion process unique among eutherian mammals. An in silico search for intact envelope genes within the Bos taurus genome identified 18 candidates belonging to five endogenous retrovirus families, with one gene displaying both placenta-specific expression, as assessed by quantitative RT-PCR analyses of a large panel of tissues, and conservation in the Ovis aries genome. Both the bovine and ovine orthologs displayed fusogenic activity by conferring infectivity on retroviral pseudotypes and triggering cell-cell fusion. In situ hybridization of placenta sections revealed specific expression in the trophoblast binucleate cells, consistent with a role in the formation--by heterologous cell fusion with uterine cells--of the trinucleate cells of the cow and the syncytial plaques of the ewe. Finally, we show that this gene, which we named "Syncytin-Rum1," is conserved among 16 representatives of higher ruminants, with evidence for purifying selection and conservation of its fusogenic properties, over 30 millions years of evolution. These data argue for syncytins being a major driving force in the emergence and diversity of the placenta.

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.