We have previously demonstrated that the envelope proteins of a murine and primate retrovirus are immunosuppressive in vivo. This property was manifested by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to have the env-expressing cells escape (at least transiently) immune rejection. Here, we analyzed the immunosuppressive activity of the human and murine syncytins. These are envelope genes from endogenous retroviruses independently coopted by ancestral hosts, conserved in evolution, specifically expressed in the placenta, and with a cell-cell fusogenic activity likely contributing to placenta morphogenesis. We show that in both humans and mice, one of the two syncytins (human syncytin-2 and mouse syncytin-B) is immunosuppressive and, rather unexpectedly, the other (human syncytin-1 and mouse syncytin-A) is not (albeit able to induce cell-cell fusion). Delineation of the immunosuppressive domain by deletion analysis, combined with a comparison between immunosuppressive and nonimmunosuppressive sequences, allowed us to derive a mutation rule targeted to specific amino acids, resulting in selective switch from immunosuppressive to nonimmunosuppressive envelope proteins and vice versa. These results unravel a critical function of retroviral envelopes, not necessarily ''individually'' selected for in the retrovirus endogenization process, albeit ''tandemly'' conserved in evolution for the syncytin pairs in primates and Muridae. Selective inactivation of immunosuppression, under conditions not affecting fusogenicity, should be important for understanding the role of this function in placental physiology and maternofetal tolerance.endogenous retrovirus ͉ fusogenicity ͉ immunosuppression ͉ HERV T he placenta is an autonomous and transient organ essentially intended for feeding and oxygenating the embryo and the fetus during intrauterine life. In several mammalian species, including Homo sapiens, the fusion of trophoblastic cells into a multinucleated layer called syncytiotrophoblast constitutes a key process of placental morphogenesis. The syncytiotrophoblast, being the main maternofetal barrier in direct contact with maternal blood, performs the essential trophic exchange functions between mother and fetus, along with the secretion of hormones and growth factors, maintenance of homeostasis, and the necessary inhibition of the mother's immune response against the allogeneic determinants of the fetus (1-3).Little is known about the molecular mechanisms involved in trophoblastic differentiation. However, a major advance has been made by the identification of envelope (Env) proteins encoded by endogenous retroviruses (ERVs) and likely involved in the formation of the syncytiotrophoblast (4-7). The human and murine genomes indeed harbor thousands of ERV elements that display a structure close to that of the integrated proviral form of exogenous retroviruses and that most probably are the remnants of past infections of the germ line by ancestral retroviruses (8-11)...
One contribution of 13 to a Theme Issue 'Paleovirology: insights from the genomic fossil record'. 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.
Endogenous retroviruses are multicopy retroelements accounting for nearly 10% of murine or human genomes. These retroelements spread into our ancestral genome millions of years ago and have acted as a driving force for genome evolution. Endogenous retroviruses may also be deleterious for their host, and have been implicated in cancers and autoimmune diseases. Most retroelements have lost replication competence because of the accumulation of inactivating mutations, but several, including some murine intracisternal A-particle (IAP) and MusD sequences, are still mobile. These elements encode a reverse transcriptase activity and move by retrotransposition, an intracellular copy-and-paste process involving an RNA intermediate. The host has developed mechanisms to silence their expression, mainly cosuppression and gene methylation. Here we identify another level of antiviral control, mediated by APOBEC3G, a member of the cytidine deaminase family that was previously shown to block HIV replication. We show that APOBEC3G markedly inhibits retrotransposition of IAP and MusD elements, and induces G-to-A hypermutations in their DNA copies. APOBEC3G, by editing viral genetic material, provides an ancestral wide cellular defence against endogenous and exogenous invaders.
Syncytins are envelope genes of retroviral origin that have been coopted 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.synepitheliochorial | placental evolution | phylogeny | placentome | ERV
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.
BackgroundSyncytins are envelope genes of retroviral origin that have been co-opted by the host to mediate a specialized function in placentation. Two of these genes have already been identified in primates, as well as two distinct, non orthologous genes in rodents.ResultsHere we identified within the rabbit Oryctolagus cuniculus-which belongs to the lagomorpha order- an envelope (env) gene of retroviral origin with the characteristic features of a bona fide syncytin, that we named syncytin-Ory1. An in silico search for full-length env genes with an uninterrupted open reading frame within the rabbit genome first identified two candidate genes that were tested for their specific expression in the placenta by quantitative RT-PCR of RNA isolated from a large set of tissues. This resulted in the identification of an env gene with placenta-specific expression and belonging to a family of endogenous retroelements present at a limited copy number in the rabbit genome. Functional characterization of the identified placenta-expressed env gene after cloning in a CMV-driven expression vector and transient transfection experiments, demonstrated both fusogenic activity in an ex vivo cell-cell fusion assay and infectivity of pseudotypes. The receptor for the rabbit syncytin-Ory1 was found to be the same as that for human syncytin-1, i.e. the previously identified ASCT2 transporter. This was demonstrated by a co-culture fusion assay between hamster A23 cells transduced with an expression vector for ASCT2 and A23 cells transduced with syncytin-Ory1. Finally, in situ hybridization of rabbit placenta sections with a syncytin-Ory1 probe revealed specific expression at the level of the junctional zone between the placental lobe and the maternal decidua, where the invading syncytial fetal tissue contacts the maternal decidua to form the labyrinth, consistent with a role in the formation of the syncytiotrophoblast. The syncytin-Ory1 gene is found in Leporidae but not in Ochotonidae, and should therefore have entered the lagomorpha order 12-30 million years ago.ConclusionThe identification of a novel syncytin gene within a third order of mammals displaying syncytiotrophoblast formation during placentation strongly supports the notion that on several occasions retroviral infections have resulted in the independent capture of genes that have been positively selected for a convergent physiological role.
An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neoRT). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) (6). The homology between proviruses and "retrotransposons" (1, 6) is further strengthened by the occurrence of intracellular "virus-like particles" and of intermediates of reverse transcription in the case of copia and Tyl (7-11).In mammals, analysis of transposition is difficult to perform by using the biological assays that have been developed in Drosophila or yeast (6,12). Accordingly, putative transposons have been suspected from their structural similarities with proviruses and their high copy number in the genome. In the case of the IAP sequences, transposition was demonstrated by the discovery of their integration into unusual loci (13-15), a situation reminiscent of the retrovirus insertions observed in a number of tumors (16).These observations raise a series of questions. First, how can it be demonstrated that putative transposons actually transpose in mammalian cells; in particular, are proviral copies of the retrovirus able to transpose? Second, is transposition modulated by genetic or epigenetic factors as observed in Drosophila and yeast (6,(17)(18)(19)? Third, can transposition be implicated in tumorigenic processes?In addressing these questions, the potentially low frequency of transposition has to be taken into account (see refs. 20 and 21 for yeast and Drosophila). To analyze transposition in mammalian cells, we therefore constructed an "indicator gene for retrotransposition," which should allow the detection of transposition (by selective procedures) for any genetic element that transposes via an RNA intermediate. MATERIALS AND METHODSPlasmid Construction. To construct the retrotransposition indicator (neo)RT, the sequence for polyadenylylation of the thymidine kinase (tk) gene of the herpes simplex virus was isolated from plasmid pAGO (22) as a Sma I/Nco I 310-basepair (bp) fragment and was inserted at the unique BamHI site of pZip plasmid (23) between the Moloney murine leukemia virus (Mo-MLV) donor and acceptor splice sites after Klenow enzyme treatment of both fragments. The fragment containing the polyadenylylation sequence and the splice sites was next isolated as a Kpn I/Kpn I fragment and inserted between the SVtk promoter and the coding region of the neo gene at the Bgl II site of pSVtkneop3 (24) after Klenow treatment of both fragments; the neoRT indicator gene (Fig. L4) was then isolated as a HindIII/Sma I fragment.To delete the env genes in pMov3 (25) The final construction [pMo-MLV(neo)RTI was obtained upon ligation of the deleted pMov3 vector with the (neo)RT indicator gene after Klenow treatment (Fig. 1B).Cell Culture, Transfection, and Infection. Methods are described in ref. 27. Cells used were NIH 3T3 and FG10 (28), and the viral producer lines 3T3 Mo-MLV for Moloney and E404B for . 3T3 LacZ cells were a gift from D. Rocancourt and C. Bonnerot (Institut Pasteur). Selection of G418-resistant cells ...
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.
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