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.     

Envelope gene of the human endogenous retrovirus HERV-W encodes a functional retrovirus envelope

A member of the human endogenous retrovirus (HERV) family termed HERV-W encodes a highly fusogenic membrane glycoprotein that appears to be expressed specifically in the placenta. It is unclear whether the glycoproteins of the HERVs can serve as functional retrovirus envelope proteins to confer infectivity on retrovirus particles. We found that the HERV-W envelope glycoprotein can form pseudotypes with human immunodeficiency virus type 1 virions and confers tropism for CD4-negative cells. Thus, the HERV-W env gene represents the first HERV env gene demonstrated to encode the functional properties of a retrovirus envelope glycoprotein.     

Synthesis, assembly, and processing of the Env ERVWE1/syncytin human endogenous retroviral envelope

Syncytin is a fusogenic protein involved in the formation of the placental syncytiotrophoblast layer. This protein is encoded by the envelope gene of the ERVWE1 proviral locus belonging to the human endogenous retrovirus W (HERV-W) family. The HERV-W infectious ancestor entered the primate lineage 25 to 40 million years ago. Although the syncytin fusion property has been clearly demonstrated, little is known about this cellular protein maturation process with respect to classical infectious retrovirus envelope proteins. Here we show that the cellular syncytin protein is synthesized as a glycosylated gPr73 precursor cleaved into two mature proteins, a gp50 surface subunit (SU) and a gp24 transmembrane subunit (TM). These SU and TM subunits are found associated as homotrimers. The intracytoplasmic tail is critical to the fusogenic phenotype, although its cleavage requirements seem to have diverged from those of classical retroviral maturation.     

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.     

A retroviral promoter and a cellular enhancer define a bipartite element which controls env ERVWE1 placental expression

The HERV-W family contains hundreds of loci diversely expressed in several physiological and pathological contexts. A unique locus termed ERVWE1 encodes an envelope glycoprotein (syncytin) involved in hominoid placental physiology. Here we show that syncytin expression is regulated by a bipartite element consisting of a cyclic AMP (cAMP)-inducible long terminal repeat (LTR) retroviral promoter adjacent to a cellular enhancer conferring a high level of expression and placental tropism. Deletion mutant analysis showed that the ERVWE1 5' LTR contains binding sites essential for basal placental activity in the region from positions +1 to +125. The region from positions +125 to +310 represents a cAMP-responsive core HERV-W promoter active in all cell types. Site-directed mutagenesis analysis highlighted the complexity of U3 regulation. ERVWE1 placenta-specific positive (e.g., T240) and negative (e.g., G71) regulatory sites were identified, as were essential sites required for basic activity (e.g., A247). The flanking sequences of the ERVWE1 provirus contain several putative regulatory elements. The upstream HERV-H and HERV-P LTRs were found to be inactive. Conversely, the 436-bp region located between the HERV-P LTR and ERVWE1 was shown to be an upstream regulatory element (URE) which is significantly active in placenta cells. This URE acts as a tissue-specific enhancer. Genetic and functional analyses of hominoid UREs revealed large differences between UREs of members of the Hominidae and the Hylobatidae. These data allowed the identification of a positive regulatory region from positions -436 to -128, a mammalian apparent LTR retrotransposon negative regulatory region from positions -128 to -67, and a trophoblast-specific enhancer (TSE) from positions -67 to -35. Putative AP-2, Sp-1, and GCMa binding sites are essential constituents of the 33-bp TSE.     

The HERV-W/7q family in the human genome. Potential for protein expression and gene regulation.

A new family of human endogenous retroviruses has recently been discovered. The best known example of a full length member of this family, HERV-W/7q, is located on chromosome 7. HERV-W/7q is characterized by a long open reading frame within its env gene which is expressed in various tissues, and mainly in placenta, as a protein that we called enverin. A search for new retroviral sequences related to the HERV-W/7q family allowed the characterization of such elements in chromosome 6. A novel full length HERV with an env gene of the HERV-W/7q type, potentially encoding a truncated form of enverin has been identified on chromosome 10. The distribution of HERV-W/7q related sequences close to or within genes offers the possibility that the expression of these genes may be regulated by their companion retroviral sequences.     

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.     

Relationship of the env genes and the endonuclease domain of the pol genes of simian foamy virus type 1 and human foamy virus.

We have molecularly cloned and sequenced a portion of the simian foamy virus type 1 (SFV-1); open reading frames representing the endonuclease domain of the polymerase (pol) and the envelope (env) genes were identified by comparison with the human foamy virus (HFV). Unlike the HFV genomic organization, the SFV-1 pol gene overlaps the env gene; thus, the open reading frames reported for HFV between pol and env is not present in SFV-1. Comparisons of predicted amino acid sequences of HFV and SFV-1 reveal that the endonuclease domains of the pol genes are about 84% related. The region predicted to encode the SFV-1 extracellular env domain is 569 codons; SFV-1 and HFV have 64% amino acid similarity in this env domain. The predicted hydrophobic transmembrane env proteins of both HFV and SFV-1 show about 73% similarity. A total of 16 potential glycosylation sites are found in SFV-1 env, and 15 are found in HFV; 11 are shared. SFV-1 has 25 cysteine residues, and HFV has 23 residues; all 23 cysteine residues of HFV are conserved in SFV-1. This sequence analysis reveals that the human and simian foamy viruses are highly related.     

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 approximately 30-million-year-old ERVPb1 envelope gene is evolutionarily conserved among hominoids and Old World monkeys

Most human endogenous retroviruses (HERVs) are ancient and their genes are rendered nonfunctional by debilitating mutations. One exception is a recently discovered envelope gene located on chromosome 14. This envelope protein was also recently shown to be expressed in various human tissues and to mediate cell-cell fusion ex vivo. In this study, we demonstrate that this locus (designated ERVPb1) is preserved in Old World monkeys and that the reading frame is maintained. This is congruent with the entry of the HERV-P(b) group between 27 and 36 million years ago as suggested by long terminal repeat divergence. Although the coding capacity is generally lost in the HERV-IP supergroup, the analysis of nucleotide substitutions, lack of stop codons, and single-nucleotide polymorephisms strongly indicates a selective advantage of the ERVPb1 envelope genes during primate evolution. The purifying selection and tissue-specific expression of the human ERVPb1 envelope gene provide strong evidence of a beneficial role for the host.     

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.     

Isolation and characterization of PERV-C env from domestic pig in Korea

Clone PERV-C (A3) env was isolated from the genomic DNA of domestic pig (Sus scrofa domesticus) in Korea to investigate the molecular properties of PERV-C. The nucleic acid homologies between the PERV-MSL (type C) reference and the PERV-C(A3) clone was 99% for env, but a single base pair deletion was found in the transmembrane (TM) region of the env open reading frame. To examine the functional characteristics of truncated PERV-C env, we constructed a replication-incompetent retroviral vector by replacing the env gene of the pCL-Eco retrovirus vector with PERV-C env. A retroviral vector bearing PERV-C/A chimeric envelopes was also created to complement the TM defect. Our results indicated that truncated PERV-C env was not infectious in human cells as expected. Interestingly, however, the vector with the PERV-C/A envelope was able to infect 293 cells. This observation suggests that recombination within PERV-C TM could render PERV-C infectious in humans. To further characterize PERV-C/A envelopes, we constructed an infectious molecular clone by using a PCR-based technique. This infectious molecular clone will be useful to examine more specific regions that are critical for human cell tropism.     

A weakly pathogenic Rauscher spleen focus-forming virus mutant that lacks the carboxyl-terminal membrane anchor of its envelope glycoprotein.

A mutant Rauscher spleen focus-forming virus (mutant 4-3) that causes mild splenic erythroblastosis in mice has a 44-base-pair deletion in the 3' region of its envelope glycoprotein (env) gene. The encoded glycoprotein terminates prematurely, lacks a hydrophobic membrane anchor, and has a shortened intracellular lifespan. An active site for causing erythroblast proliferation may occur in the undamaged amino-terminal domain of the env glycoprotein.     

Sequence and expression of the mouse mammary tumour virus env gene

We have determined the DNA sequence of the envelope gene region of the GR strain of mouse mammary tumour virus. The sequence extends for 3012 nucleotides from the single EcoRI site to beyond the PstI site in the 3' long terminal repeat (LTR) of the provirus. There is a major open reading frame from nucleotides 752 to 2818 which encompasses the entire env gene. This reading frame extends through a polypurine tract and into the LTR. There is another open reading frame from the first nucleotide to position 803, presumably corresponding to the end of the pol gene. The splice acceptor site which generates env mRNA has been mapped experimentally to nucleotide 750. The env gene products, gp52 and gp36, have been positioned on the sequence using the directly determined amino acid sequences of the amino terminus of gp52; and both the amino and carboxyl termini of gp36. The start of gp52 is preceded by a series of 19 uncharged amino acids which could function as a typical signal sequence, but this sequence is only part of a much longer leader peptide. The tetrad Arg-Ala-Lys-Arg is the presumed cleavage site in the gPr73env precursor, and occurs just before the gp36 amino terminus. There are five potential asparagine-linked glycosylation sites which agrees with previous experimental results. The gp36 has two long hydrophobic regions at its amino and carboxy termini, these are suggested to act as a fusion peptide and the trans-membrane anchor, respectively.     

Regulation of human immunodeficiency virus env expression by the rev gene product.

A single simian virus 40 late replacement vector which expresses both the rev and envelope (env) genes of human immunodeficiency virus was used to examine the mechanism underlying the dependence of env gene expression on the rev protein. When rev was deleted from the vector, no envelope protein expression could be detected in transfected cells, and the levels of cytoplasmic env mRNA were dramatically reduced. In contrast to this, the levels of env RNA in total cellular RNA preparations were similar with or without rev coexpression, and analysis of nuclear RNA showed that the levels of nuclear env RNA were increased in the absence of rev. These results suggest that rev functions to regulate nuclear export of env mRNA. It was possible to restore env expression from the vector lacking rev by supplying rev in trans, provided that a cis-acting sequence was also present. This sequence was mapped to a 854-base-pair region within the env open reading frame, and it was shown that the sequence could be moved but that it worked only in its original orientation.     

Comparison of the entire genomes of bovine leukemia virus and human T-cell leukemia virus and characterization of their unidentified open reading frames.

We have compared the sequence of the entire genomes of bovine leukemia virus (BLV) and human T-cell leukemia virus type I (HTLV-I). Both the gag and pol genes show overall strong homologies indicating the close evolutionary relationship of the two retroviruses. However, a surface glycoprotein portion of the env gene shows no appreciable homology, which probably reflects a difference in their host ranges. The 3' end portion of the BLV genome (designated as pXBL) contains an unidentified long open reading frame that has a typical protein-coding property. The potential product of this open reading frame may be a glycoprotein of approximately 40 000 daltons. We note that its amino acid sequence shows low but appreciable homology, especially in its N-terminal quarter, to that of the HTLV-I counterpart (pX product), and we thus suggest that BLV pXBL and HTLV-I pX have diverged from a common ancestral gene. It is tentatively concluded that both the putative pXBL and pX products are respectively produced from a spliced mRNA.