RESUMO
Detection of nucleic acids and induction of type I interferons (IFNs) are principal elements of antiviral defense but can cause autoimmunity if misregulated. Cytosolic DNA detection activates a potent, cell-intrinsic antiviral response through a poorly defined pathway. In a screen for proteins relevant to this IFN-stimulatory DNA (ISD) response, we identify 3' repair exonuclease 1 (Trex1). Mutations in the human trex1 gene cause Aicardi-Goutieres syndrome (AGS) and chilblain lupus, but the molecular basis of these diseases is unknown. We define Trex1 as an essential negative regulator of the ISD response and delineate the genetic pathway linking Trex1 deficiency to lethal autoimmunity. We show that single-stranded DNA derived from endogenous retroelements accumulates in Trex1-deficient cells, and that Trex1 can metabolize reverse-transcribed DNA. These findings reveal a cell-intrinsic mechanism for initiation of autoimmunity, implicate the ISD pathway as the cause of AGS, and suggest an unanticipated contribution of endogenous retroelements to autoimmunity.
Assuntos
Autoimunidade , DNA de Cadeia Simples/imunologia , Exodesoxirribonucleases/imunologia , Fosfoproteínas/imunologia , Retroelementos/genética , Animais , Autoanticorpos/imunologia , Linhagem Celular , Exodesoxirribonucleases/genética , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Camundongos , Fosfoproteínas/genética , Retroelementos/imunologiaRESUMO
Approximately 10% of the mouse genome is composed of endogenous retroviruses belonging to different families. In contrast to the situation in the human genome, several of these families correspond to recent, still-infectious elements capable of encoding complete viral particles. The mouse GLN endogenous retrovirus is one of these active families. We previously identified one fully functional provirus from the sequenced genome of the C57BL/6 mouse strain. The GLN envelope protein gives the infectious viral particles an ecotropic host range, and we had demonstrated that the receptor was neither CAT1 nor SMIT1, the two previously identified receptors for mouse ecotropic retroviral envelope proteins. In this study, we have identified SLC19A1, the reduced folate carrier, as the cellular protein used as a receptor by the GLN retrovirus. The ecotropic tropism exhibited by this envelope is due to the presence or absence of an N-linked glycosylation site in the first extracellular loop as well as the specific amino acid sequence of the extracellular domains of the receptor. Like all the other retroviral envelope proteins from the gammaretrovirus genus whose receptors have been identified, the GLN envelope protein uses a member of the solute carrier superfamily as a receptor.IMPORTANCE Endogenous retroviruses are genomic traces of past infections present in all vertebrates. Most of these elements degenerate over time and become nonfunctional, but the mouse genome still contains several families with full infection abilities. The GLN retrovirus is one of them, and its members encode particles that are able to infect only mouse cells. Here, we identified the cellular protein used as a receptor by GLN for cell entry. It is SLC19A1, the reduced folate carrier. We show that GLN infection is limited to mouse cells due to both a mutation in the mouse gene preventing the glycosylation of SLC19A1 and also other residues conserved within the rat but not in the hamster and human proteins. Like all other gammaretroviruses whose receptors have been identified, GLN uses a member of the solute carrier superfamily for cell entry, highlighting the role of these proteins for retroviral infection in mammals.
Assuntos
Gammaretrovirus/metabolismo , Produtos do Gene env/genética , Receptores Virais/genética , Proteína Carregadora de Folato Reduzido/genética , Proteínas do Envelope Viral/genética , Ligação Viral , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Gammaretrovirus/genética , Genoma/genética , Glicosilação , Células HEK293 , Especificidade de Hospedeiro , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Proteína Carregadora de Folato Reduzido/metabolismo , Infecções por Retroviridae/virologiaRESUMO
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.
Assuntos
Retrovirus Endógenos/genética , Hyaenidae/genética , Hyaenidae/virologia , Sequência de Aminoácidos , Animais , Gatos , Cães , Feminino , Perfilação da Expressão Gênica/métodos , Genes env/genética , Filogenia , Placenta/virologia , Gravidez , Retroviridae/genética , Análise de Sequência de RNA/métodos , Proteínas do Envelope Viral/genéticaRESUMO
Capture of retroviral envelope genes is likely to have played a role in the emergence of placental mammals, with evidence for multiple, reiterated, and independent capture events occurring in mammals, and be responsible for the diversity of present day placental structures. Here, we uncover a full-length endogenous retrovirus envelope protein, dubbed HEMO [human endogenous MER34 (medium-reiteration-frequency-family-34) ORF], with unprecedented characteristics, because it is actively shed in the blood circulation in humans via specific cleavage of the precursor envelope protein upstream of the transmembrane domain. At variance with previously identified retroviral envelope genes, its encoding gene is found to be transcribed from a unique CpG-rich promoter not related to a retroviral LTR, with sites of expression including the placenta as well as other tissues and rather unexpectedly, stem cells as well as reprogrammed induced pluripotent stem cells (iPSCs), where the protein can also be detected. We provide evidence that the associated retroviral capture event most probably occurred >100 Mya before the split of Laurasiatheria and Euarchontoglires, with the identified retroviral envelope gene encoding a full-length protein in all simians under purifying selection and with similar shedding capacity. Finally, a comprehensive screen of the expression of the gene discloses high transcript levels in several tumor tissues, such as germ cell, breast, and ovarian tumors, with in the latter case, evidence for a histotype dependence and specific protein expression in clear-cell carcinoma. Altogether, the identified protein could constitute a "stemness marker" of the normal cell and a possible target for immunotherapeutic approaches in tumors.
Assuntos
Retrovirus Endógenos/metabolismo , Neoplasias/metabolismo , Placenta/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas do Envelope Viral/biossíntese , Adulto , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Proteínas de Neoplasias , Neoplasias/virologia , Placenta/virologia , Células-Tronco Pluripotentes/virologia , GravidezRESUMO
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.
Assuntos
Proteínas de Transporte/genética , Retrovirus Endógenos/genética , Produtos do Gene env/genética , Lagartos/genética , Placenta/metabolismo , Proteínas da Gravidez/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Retrovirus Endógenos/metabolismo , Evolução Molecular , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Produtos do Gene env/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Imuno-Histoquímica , Lagartos/metabolismo , Filogenia , Gravidez , Proteínas da Gravidez/metabolismo , Proteínas do Envelope Viral/genéticaRESUMO
Endogenous retroviruses are cellular genes of retroviral origin captured by their host during the course of evolution and represent around 8% of the human genome. Although most are defective and transcriptionally silenced, some are still able to generate retroviral-like particles and proteins. Among these, the HERV-K(HML2) family is remarkable since its members have amplified relatively recently and many of them still have full length coding genes. Furthermore, they are induced in cancers, especially in melanoma, breast cancer and germ cell tumours, where viral particles, as well as the envelope protein (Env), can be detected. Here we show that HERV-K(HML2) Env per se has oncogenic properties. Its expression in a non-tumourigenic human breast epithelial cell line induces epithelial to mesenchymal transition (EMT), often associated with tumour aggressiveness and metastasis. In our model, this is typified by key modifications in a set of molecular markers, changes in cell morphology and enhanced cell motility. Remarkably, microarrays performed in 293T cells reveal that HERV-K(HML2) Env is a strong inducer of several transcription factors, namely ETV4, ETV5 and EGR1, which are downstream effectors of the MAPK ERK1/2 and are associated with cellular transformation. We demonstrate that HERV-K(HML2) Env effectively activates the ERK1/2 pathway in our experimental setting and that this activation depends on the Env cytoplasmic tail. In addition, this phenomenon is very specific, being absent with every other retroviral Env tested, except for Jaagsiekte Sheep Retrovirus (JSRV) Env, which is already known to have transforming properties in vivo. Though HERV-K Env is not directly transforming by itself, the newly discovered properties of this protein may contribute to oncogenesis.
Assuntos
Retrovirus Endógenos/genética , Transição Epitelial-Mesenquimal/genética , Sistema de Sinalização das MAP Quinases/genética , Animais , Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Produtos do Gene env/genética , Humanos , Retrovirus Jaagsiekte de Ovinos , Invasividade Neoplásica , Ovinos/genéticaRESUMO
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.
Assuntos
Produtos do Gene env/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteínas da Gravidez/genética , Animais , Diferenciação Celular/genética , Cães , Retrovirus Endógenos/genética , Feminino , Técnicas de Inativação de Genes , Produtos do Gene env/metabolismo , Humanos , Masculino , Mamíferos , Camundongos , Músculo Esquelético/crescimento & desenvolvimento , Proteínas da Gravidez/metabolismo , RNA Interferente Pequeno/genética , Regeneração/genética , Caracteres SexuaisRESUMO
Retroviral integration into germline DNA can result in the formation of a vertically inherited proviral sequence called an endogenous retrovirus (ERV). Over the course of their evolution, vertebrate genomes have accumulated many thousands of ERV loci. These sequences provide useful retrospective information about ancient retroviruses, and have also played an important role in shaping the evolution of vertebrate genomes. There is an immediate need for a unified system of nomenclature for ERV loci, not only to assist genome annotation, but also to facilitate research on ERVs and their impact on genome biology and evolution. In this review, we examine how ERV nomenclatures have developed, and consider the possibilities for the implementation of a systematic approach for naming ERV loci. We propose that such a nomenclature should not only provide unique identifiers for individual loci, but also denote orthologous relationships between ERVs in different species. In addition, we propose that-where possible-mnemonic links to previous, well-established names for ERV loci and groups should be retained. We show how this approach can be applied and integrated into existing taxonomic and nomenclature schemes for retroviruses, ERVs and transposable elements.
Assuntos
Retrovirus Endógenos/classificação , Retrovirus Endógenos/genética , Animais , Evolução Molecular , Loci Gênicos , Variação Genética , Genômica , Humanos , Terminologia como Assunto , Vertebrados/genética , Vertebrados/virologiaRESUMO
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.
Assuntos
Antígenos Ly/metabolismo , Fusão Celular , Proteínas da Gravidez/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Antígenos Ly/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Testes Genéticos/métodos , Camundongos , Receptores de Superfície Celular/genéticaRESUMO
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 â¼190 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.
Assuntos
Produtos do Gene env/genética , Marsupiais/genética , Placenta/fisiologia , Proteínas da Gravidez/genética , Retroviridae/fisiologia , Proteínas do Envelope Viral/genética , Animais , Feminino , Perfilação da Expressão Gênica , Genes env , Hibridização In Situ , Marsupiais/classificação , Dados de Sequência Molecular , Filogenia , Gravidez , Transcrição GênicaRESUMO
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.
Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Tatus/virologia , Betaretrovirus/isolamento & purificação , Retrovirus Endógenos/isolamento & purificação , Antígenos de Histocompatibilidade Menor/metabolismo , Provírus/isolamento & purificação , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Betaretrovirus/genética , Retrovirus Endógenos/genética , Testes Genéticos , Provírus/genética , Recombinação Genética , Proteínas do Envelope Viral/genéticaRESUMO
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.
Assuntos
Eulipotyphla/genética , Produtos do Gene env/genética , Filogenia , Placentação/genética , Proteínas da Gravidez/genética , Retroviridae/genética , Animais , Simulação por Computador , Evolução Molecular , Feminino , Genoma/genética , Hibridização In Situ , Dados de Sequência Molecular , Placenta/citologia , Placenta/ultraestrutura , Gravidez , Provírus/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Seleção Genética , Fatores de Tempo , Integração Viral/genéticaRESUMO
Long non-coding RNAs (lncRNAs) are widely expressed and play various roles in cell homeostasis. However, because of their low conservation at the sequence level, recapitulating lncRNA evolutionary history is often challenging. While performing an ultrastructural analysis of viral particles present in uterine glands of gestating opossum females, we serendipitously noticed the presence of numerous structures similar to paraspeckles, nuclear bodies which in human and mouse cells are assembled around an architectural NEAT1/MENϵ/ß lncRNA. Here, using an opossum kidney (OK) cell line, we confirmed by immuno-electron microscopy the presence of paraspeckles in marsupials. We then identified the orthologous opossum NEAT1 gene which, although poorly conserved at the sequence level, displays NEAT1 characteristic features such as short and long isoforms expressed from a unique promoter and for the latter an RNase P cleavage site at its 3'-end. Combining tissue-specific qRT-PCR, in situ hybridization at the optical and electron microscopic levels, we show that (i) NEAT1 is paraspeckle-associated in opossum (ii) NEAT1 expression is strongly induced in late gestation in uterine/placental extracts (iii) NEAT1 induction occurs in the uterine gland nuclei in which paraspeckles were detected. Finally, treatment of OK cells with proteasome inhibitors induces paraspeckle assembly, as previously observed in human cells. Altogether, these results demonstrate that paraspeckles are tissue-specific, stress-responding nuclear bodies in marsupials, illustrating their structural and functional continuity over 200 My of evolution throughout the mammalian lineage. In contrast, the rapid evolution of the NEAT1 transcripts highlights the relaxed constraint that, despite functional conservation, is exerted on this lncRNA.
Assuntos
Evolução Molecular , Monodelphis/genética , RNA Longo não Codificante/genética , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Mapeamento Cromossômico , Expressão Gênica , Conformação de Ácido Nucleico , Especificidade de Órgãos/genética , Organogênese/genética , Isoformas de RNA , RNA Longo não Codificante/químicaRESUMO
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.
Assuntos
Evolução Biológica , Produtos do Gene env , Placentação , Proteínas da Gravidez , Proteínas dos Retroviridae , Animais , Cercopithecidae/genética , Retrovirus Endógenos , Feminino , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene env/fisiologia , Genoma , Humanos , Filogenia , Placenta/fisiologia , Placentação/genética , Placentação/fisiologia , Gravidez , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Proteínas da Gravidez/fisiologia , Primatas/genética , Proteínas dos Retroviridae/genética , Proteínas dos Retroviridae/metabolismoRESUMO
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.
Assuntos
Retrovirus Endógenos/genética , Produtos do Gene env/genética , Cabras/genética , Placenta/anatomia & histologia , Proteínas da Gravidez/genética , Ruminantes/genética , Proteínas do Envelope Viral/genética , Animais , Bovinos , Biologia Computacional , Sequência Conservada , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Estudos de Associação Genética , Variação Genética , Genoma/genética , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Filogenia , Placenta/citologia , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Seleção Genética , Transcrição GênicaRESUMO
UNLABELLED: Endogenous retroviruses are the remnants of past retroviral infections that are scattered within mammalian genomes. In humans, most of these elements are old degenerate sequences that have lost their coding properties. The HERV-K(HML2) family is an exception: it recently amplified in the human genome and corresponds to the most active proviruses, with some intact open reading frames and the potential to encode viral particles. Here, using a reconstructed consensus element, we show that HERV-K(HML2) proviruses are able to inhibit Tetherin, a cellular restriction factor that is active against most enveloped viruses and acts by keeping the viral particles attached to the cell surface. More precisely, we identify the Envelope protein (Env) as the viral effector active against Tetherin. Through immunoprecipitation experiments, we show that the recognition of Tetherin is mediated by the surface subunit of Env. Similar to Ebola glycoprotein, HERV-K(HML2) Env does not mediate Tetherin degradation or cell surface removal; therefore, it uses a yet-undescribed mechanism to inactivate Tetherin. We also assessed all natural complete alleles of endogenous HERV-K(HML2) Env described to date for their ability to inhibit Tetherin and found that two of them (out of six) can block Tetherin restriction. However, due to their recent amplification, HERV-K(HML2) elements are extremely polymorphic in the human population, and it is likely that individuals will not all possess the same anti-Tetherin potential. Because of Tetherin's role as a restriction factor capable of inducing innate immune responses, this could have functional consequences for individual responses to infection. IMPORTANCE: Tetherin, a cellular protein initially characterized for its role against HIV-1, has been proven to counteract numerous enveloped viruses. It blocks the release of viral particles from producer cells, keeping them tethered to the cell surface. Several viruses have developed strategies to inhibit Tetherin activity, allowing them to efficiently infect and replicate in their host. Here, we show that human HERV-K(HML2) elements, the remnants of an ancient retroviral infection, possess an anti-Tetherin activity which is mediated by the envelope protein. It is likely that this activity was an important factor that contributed to the recent, human-specific amplification of this family of elements. Also, due to their recent amplification, HERV-K(HML2) elements are highly polymorphic in the human population. Since Tetherin is a mediator of innate immunity, interindividual variations among HERV-K(HML2) Env genes may result in differences in immune responses to infection.
Assuntos
Antígenos CD/imunologia , Retrovirus Endógenos/imunologia , Proteínas do Envelope Viral/metabolismo , Linhagem Celular , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/imunologia , Humanos , ImunoprecipitaçãoRESUMO
We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.
Assuntos
Vírus da Varíola dos Canários/genética , Produtos do Gene env/química , Produtos do Gene env/imunologia , Imunossupressores/química , Vírus da Leucemia Felina/genética , Leucemia Felina/imunologia , Mutação de Sentido Incorreto , Proteínas Oncogênicas de Retroviridae/genética , Vacinas Virais/genética , Animais , Vírus da Varíola dos Canários/metabolismo , Gatos , Feminino , Produtos do Gene env/administração & dosagem , Produtos do Gene env/genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Imunossupressores/administração & dosagem , Imunossupressores/imunologia , Interferons/genética , Interferons/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Vírus da Leucemia Felina/química , Vírus da Leucemia Felina/imunologia , Leucemia Felina/prevenção & controle , Leucemia Felina/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Proteínas Oncogênicas de Retroviridae/administração & dosagem , Proteínas Oncogênicas de Retroviridae/química , Proteínas Oncogênicas de Retroviridae/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/química , Vacinas Virais/imunologiaRESUMO
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.
Assuntos
Retrovirus Endógenos/genética , Produtos do Gene env/genética , Placentação , Proteínas da Gravidez/genética , Sciuridae/fisiologia , Sciuridae/virologia , Animais , Sequência Conservada , Feminino , Perfilação da Expressão Gênica , Produtos do Gene env/biossíntese , Hibridização In Situ , Dados de Sequência Molecular , Gravidez , Proteínas da Gravidez/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Sciuridae/genética , Análise de Sequência de DNARESUMO
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.
Assuntos
Retrovirus Endógenos/genética , Produtos do Gene env/fisiologia , Placentação/fisiologia , Proteínas da Gravidez/fisiologia , Proteínas do Envelope Viral/fisiologia , Sequência de Aminoácidos , Animais , Carnívoros , Feminino , Dados de Sequência Molecular , Filogenia , Gravidez , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/químicaRESUMO
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.