An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor

A new human endogenous retrovirus (HERV) family, termed HERV-W, was recently described (J.-L. Blond, F. Besème, L. Duret, O. Bouton, F. Bedin, H. Perron, B. Mandrand, and F. Mallet, J. Virol. 73:1175-1185, 1999). HERV-W mRNAs were found to be specifically expressed in placenta cells, and an env cDNA containing a complete open reading frame was recovered. In cell-cell fusion assays, we demonstrate here that the product of the HERV-W env gene is a highly fusogenic membrane glycoprotein. Transfection of an HERV-W Env expression vector in a panel of cell lines derived from different species resulted in formation of syncytia in primate and pig cells upon interaction with the type D mammalian retrovirus receptor. Moreover, envelope glycoproteins encoded by HERV-W were specifically detected in placenta cells, suggesting that they may play a physiological role during pregnancy and placenta formation.     

Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians

A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians-from New World monkeys to humans-are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation-located in the TM subunit-was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution.     

The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors

The human endogenous retrovirus type W (HERV-W) family includes proviruses with intact protein-coding regions that appear to be under selection pressure, suggesting that some HERV-W proviruses may remain active in higher primates. The envelope glycoprotein (Env) encoded by HERV-W is highly fusogenic, is naturally expressed in human placental syncytiatrophoblasts, and has been reported to function as a superantigen in lymphocyte cultures. Recent evidence suggested that HERV-W Env can mediate syncytium formation by interacting with the human sodium-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) (J.-L. Blond, D. Lavillette, V. Cheynet, O. Bouton, G. Oriol, S. Chapel-Fernandez, B. Mandrand, F. Mallet, and F.-L. Cosset, J. Virol. 74:3321-3329, 2000) and that it can pseudotype human immunodeficiency virus cores (D. S. An, Y. Xie, and I. S. Y. Chen, J. Virol. 75:3488-3489, 2001). By using cell-cell fusion and pseudotype virion infection assays, we found that HERV-W Env efficiently uses both hASCT2 and the related transporter hASCT1 (gene name, SLC1A4) as receptors. In addition, although HERV-W Env mediates only slight syncytium formation or infection of mouse cells, it utilizes the mouse transporters mASCT1 and mASCT2 when their sites for N-linked glycosylation are eliminated by mutagenesis. Consistent with their role as a battlefield in host-virus coevolution, the viral recognition regions in ASCT1 and ASCT2 of humans and mice are highly divergent compared with other regions of these proteins, and their ratios of nonsynonymous to synonymous nucleotide sequence changes are extremely large. The recognition of ASCT1 and ASCT2 despite this divergence of their sequences strongly suggests that the use of both receptors has been highly advantageous for survival and evolution of the HERV-W family of retroviruses.     

Evidence of selection on the domesticated ERVWE1 env retroviral element involved in placentation

The human endogenous retrovirus HERV-W multicopy family includes a unique proviral locus, termed ERVWE1, which contains gag and pol pseudogenes and has retained a full-length envelope open reading frame (ORF). This Env protein (syncytin) is a highly fusogenic membrane glycoprotein and has been proposed to be involved in hominoid placental physiology. To track the hallmarks of natural selection acting on the ERVWE1 env gene, the pattern of substitutions and indels was analyzed within all human HERV-W elements and along the ERVWE1 orthologous loci in chimpanzee, gorilla, orangutan, and gibbon. The comparison of ERVWE1 and paralogous HERV-W copies revealed an ERVWE1-specific signature consisting of a four amino acid deletion in the intracytoplasmic tail of the glycoprotein. We show that this deletion is crucial for the envelope fusogenic activity. The comparison of the human ERVWE1 locus with its orthologs demonstrates the existence of a selective pressure to maintain the env reading frame open. Notably, the 3' part of the env gene, encoding regions required for the fusion process, is under purifying selection. The identification of selective constraints on env ERVWE1 confirms that this retroviral locus has been recruited in the hominoid lineage to become a bona fide gene.     

Molecular phylogeny and evolution of the human endogenous retrovirus HERV-W LTR family in hominoid primates

Long terminal repeats (LTRs) of human endogenous retrovirus (HERV) have contributed to the structural change or genetic variation of primate genome that are connected to speciation and evolution. Using genomic DNAs that were derived from hominoid primates (chimpanzee, gorilla, orangutan, and gibbon), we performed PCR amplification and identified thirty HERV-W LTR elements. These LTR elements showed a 82-98% sequence similarity with HERV-W LTR (AF072500). Specifically, additional sequences (GCCACCACCACTGTTT in the gorilla and TGCTGCTGACTCCCATCC in the gibbon) were noticed. Clone OR3 from the orangutan and clone GI2 from the gibbon showed a 100% sequence similarity, although they are different species. This indicates that both LTR elements were proliferated during the last 2 to 5 million years from the integration of the original LTR element. A phylogenetic tree that was obtained by the neighbor-joining method revealed a wide overlap of the LTR elements across species, suggesting that the HERV-W LTR family evolved independently during the hominoid evolution.     

Direct involvement of HERV-W Env glycoprotein in human trophoblast cell fusion and differentiation

We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation.     

Human leukemia antigen-A~*0201-restricted epitopes of human endogenous retrovirus W family envelope(HERV-W env) induce strong cytotoxic T lymphocyte responses

Human endogenous retrovirus W family(HERV-W) envelope(env) has been reported to be related to several human diseases, including autoimmune disorders, and it could activate innate immunity.However, there are no reports investigating whether human leukemia antigen(HLA)-A~*0201~+restriction is involved in the immune response caused by HERV-W env in neuropsychiatric diseases. In the present study, HERV-W env-derived epitopes presented by HLA-A~*0201 are described with the potential for use in adoptive immunotherapy. Five peptides displaying HLAA~*0201-binding motifs were predicted using SYFEPITHI and BIMAS, and synthesized. A CCK-8 assay showed peptides W, Q and T promoted lymphocyte proliferation. Stimulation of peripheral blood mononuclear cells from HLA-A~*0201~+ donors with each of these peptides induced peptidespecific CD8~+ T cells. High numbers of IFN-γ-secreting T cells were also detectable after several weekly stimulations with W, Q and T. Besides lysis of HERV-W env-loaded target cells, specific apoptosis was also observed. These data demonstrate that human T cells can be sensitized toward HERV-W env peptides(W, Q and T) and, moreover, pose a high killing potential toward HERV-W env-expressing U251 cells. In conclusion, peptides W Q and T, which are HERV-W env antigenic epitopes, have both antigenicity and immunogenicity, and can cause strong T cell immune responses. Our data strengthen the view that HERV-W env should be considered as an autoantigen that can induce autoimmunity in neuropsychiatric diseases, such as multiple sclerosis and schizophrenia. These data might provide an experimental foundation for a HERV-W env peptide vaccine and new insight into the treatment of neuropsychiatric diseases.     

Placenta-specific INSL4 expression is mediated by a human endogenous retrovirus element

The human insulin-family genes regulate cell growth, metabolism, and tissue-specific functions. Among these different members, only INSL4 gene shows a predominant placenta-specific expression. Here, we show that the human INSL4 gene is tightly clustered with three other members of the human insulin superfamily (RLN1, RLN2, and INSL6) within a 176-kilobase genomic segment on chromosome region 9p23.3-p24.1. We also report evidence that INSL4 is probably the only insulin-like growth factor gene to be primate-specific. We identified an unexpected human endogenous retrovirus (HERV) element inserted into the human INSL4 promoter with a sequence similar to that of env gene, flanked by two long terminal repeats(LTRs). The emergence of INSL4 gene and genomic insertion of HERV appear to have occurred after the divergence of New World and Old World monkeys ( approximately 45 million years ago). Transient transfection experiments showed that the placenta-specific expression of INSL4 is mediated by the 3' LTR of the HERV element, and that the latter may have a major role in INSL4 up-regulation during human cytotrophoblast differentiation into syncytiotrophoblast. Finally, we identified an INSL4 alternatively spliced mRNA species that encodes putative novel INSL4-like peptides. These data support the view that ancient retroviral infection may have been a major event in primate evolution, especially in the functional evolution of the human placenta.     

Functional characterization of the human placental fusogenic membrane protein syncytin 2

Fusion of cytotrophoblasts into the multinucleated syncytiotrophoblast layer is essential for the development of a functional placenta. The envelope protein of a human endogenous retrovirus W (HERV-W) family member, syncytin 1, has been shown to mediate placental cell fusion. Recently, the envelope protein of another HERV family member (HERV-FRD), syncytin 2, has been identified and shown to be highly expressed in the placenta. To better understand the biology of syncytin 2, in this study we first investigated syncytin 2 gene expression in normal and preeclamptic placentas and then characterized the functions of syncytin 2. The expression of syncytin 2 gene was decreased in preeclamptic placentas and could be stimulated by the cAMP stimulant forskolin. The endoprotease furin was found to be involved in the posttranslational cleavage of syncytin 1 and 2 polypeptides into surface and transmembrane subunits. In addition, proper association of the subunits of syncytins 1 and 2 is probably required for the functional integrity of each protein, because subunit swapping of syncytins 1 and 2 failed to generate fusogenic chimeras. Finally, we demonstrated that the disulfide bridge-forming CX(2)C and CX(7)C motifs found in syncytins 1 and 2 are essential for their fusogenic activities, because mutations in the CX(2)C motif not only abolished fusogenesis but also functioned as dominant-negative mutants. Our results suggest that syncytin 2 may function as a second fusogenic protein for placental cell fusion.     

Expression profiles of human endogenous retrovirus (HERV)-K and HERV-R Env proteins in various cancers

The vertebrate genome contains an endogenous retrovirus that has been inherited from the past millions of years. Although approximately 8% of human chromosomal DNA consists of sequences derived from human endogenous retrovirus (HERV) fragments, most of the HERVs are currently inactive and non-infectious due to recombination, deletions, and mutations after insertion into the host genome. Several studies suggested that Human endogenous retroviruses (HERVs) factors are significantly related to certain cancers. However, only limited studies have been conducted to analyze the expression of HERV derived elements at protein levels in certain cancers. Herein, we analyzed the expression profiles of HERV-K envelope (Env) and HERV-R Env proteins in eleven different kinds of cancer tissues. Furthermore, the expression patterns of both protein and correlation with various clinical data in each tissue were analyzed. The expressions of both HERV-K Env and HERV-R Env protein were identified to be significantly high in most of the tumors compared with normal surrounding tissues. Correlations between HERV Env expressions and clinical investigations varied depending on the HERV types and cancers. Overall expression patterns of HERV-K Env and HERV-R Env proteins were different in every individual but a similar pattern of expressions was observed in the same individual. These results demonstrate the expression profiles of HERV-K and HERV-R Env proteins in various cancer tissues and provide a good reference for the association of endogenous retroviral Env proteins in the progression of various cancers. Furthermore, the results elucidate the relationship between HERV-Env expression and the clinical significance of certain cancers.     

人内源性逆转录病毒W家族与疾病相关性的研究

人内源性逆转录病毒(human endogenous retroviruses,HERV)是几百万年前整合至人类基因组并遗传至今的外源性逆转录病毒的残余物。因突变、缺失等导致大多数HERV没有完整的开放读码框,但仍有部分家族成员可编码完整的病毒蛋白,如分离自多发性硬化症患者的γ逆转录病毒相似元件家族成员HERV-W的包膜蛋白基因(HERV-W env,又称ERVWE1)编码的ENV蛋白(又称Syncytin-1),在人胎盘发育过程中起细胞融合以及免疫调节作用。在生理条件下,HERV-W受到表观遗传调控而其转录活性被抑制;但亦可被环境、遗传等因素激活,如自身免疫性疾病、精神疾病及癌症等。研究发现HERV-W可能在疾病的发生、发展中起重要的“桥梁”与“触发”作用,靶向Syncytin-1的单克隆抗体GNbAC1已用于多发性硬化症的临床研究,并且在1型糖尿病中也有良好的应用前景。对HERV-W的深入研究可为某些疾病的诊断和治疗提供重要的途径。     

Characterization of the intragenomic spread of the human endogenous retrovirus family HERV-W

This study examines the intragenomic spread of the human endogenous retrovirus family HERV-W from insertions present within the draft sequence of the human genome. Identification of shared diagnostic differences and phylogenetic analyses revealed the existence of three main subfamilies. The average divergence between sequences for each of the subfamilies suggests that most of the HERV-W elements were inserted within the genome during a short period of evolutionary time. Each one of the subfamilies consists of two types of insertions, the expected proviral sequences and other sequences resembling the structure of processed retrogenes. These HERV-W retrosequences extend from the R region of the 5' long-terminal repeat (LTR) to the R region of the 3' LTR (as viral genomic RNAs), end in poly(A) 3' tails, and are flanked by direct repeats longer than the proviral integrations. Furthermore, several of the HERV-W retrosequences are 5'-truncated at different sites. I suggest the involvement of the L1 machinery in these integrations and discuss the characteristic features of the evolutionary history of HERV-W, with emphasis on the putative impact of HERV-W retrosequence integrations on the mammalian genome.