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.     

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.     

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.     

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.     

Human endogenous retrovirus W env increases nitric oxide production and enhances the migration ability of microglia by regulating the expression of inducible nitric oxide synthase

Human endogenous retrovirus W env(HERV-W env) plays a critical role in many neuropsychological diseases such as schizophrenia and multiple sclerosis(MS). These diseases are accompanied by immunological reactions in the central nervous system(CNS). Microglia are important immunocytes in brain inflammation that can produce a gasotransmitter – nitric oxide(NO). NO not only plays a role in the function of neuronal cells but also participates in the pathogenesis of various neuropsychological diseases. In this study, we reported increased NO production in CHME-5 microglia cells after they were transfected with HERV-W env. Moreover, HERV-W env increased the expression and function of human inducible nitric oxide synthase(hi NOS) and enhanced the promoter activity of hi NOS. Microglial migration was also enhanced. These data revealed that HERV-W env might contribute to increase NO production and microglial migration ability in neuropsychological disorders by regulating the expression of inducible NOS. Results from this study might lead to the identification of novel targets for the treatment of neuropsychological diseases, including neuroinflammatory diseases, stroke, and neurodegenerative diseases.     

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.     

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.     

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。     

Molecular characterization of the HERV-W env gene in humans and primates: expression, FISH, phylogeny, and evolution

We characterized the human endogenous retrovirus (HERV-W) family in humans and primates. In silico expression data indicated that 22 complete HERV-W families from human chromosomes 1-3, 5-8, 10-12, 15, 19, and X are randomly expressed in various tissues. Quantitative real-time RT-PCR analysis of the HERV-W env gene derived from human chromosome 7q21.2 indicated predominant expression in the human placenta. Several copies of repeat sequences (SINE, LINE, LTR, simple repeat) were detected within the complete or processed pseudo HERV-W of the human, chimpanzee, and rhesus monkey. Compared to other regions (5'LTR, Gag, Gag-Pol, Env, 3'LTR), the repeat family has been mainly integrated into the region spanning the 5'LTRs of Gag (1398 bp) and Pol (3242 bp). FISH detected the HERV-W probe (fosWE1) derived from a gorilla fosmid library in the metaphase chromosomes of all primates (five hominoids, three Old World monkeys, two New World monkeys, and one prosimian), but not in Tupaia. This finding was supported by molecular clock and phylogeny data using the divergence values of the complete HERV-W LTR elements. The data suggested that the HERV-W family was integrated into the primate genome approximately 63 million years (Myr) ago, and evolved independently during the course of primate radiation.     

Nucleotide sequence of human endogenous retrovirus genome related to the mouse mammary tumor virus genome.

We determined the complete nucleotide sequence of the human endogenous retrovirus genome HERV-K10 isolated as the sequence homologous to the Syrian hamster intracisternal A-particle (type A retrovirus) genome. HERV-K10 is 9,179 base pairs long with long terminal repeats of 968 base pairs at both ends; a sequence 290 base pairs long, however, was found to be deleted. It was concluded that a composite genome having the 290-base-pair fragment is the prototype HERV-K provirus gag (666 codons), protease (334 codons), pol (937 codons), and env (618 codons) genes. The size of the protease gene product of HERV-K is essentially the same as that of A- and D-type oncoviruses but nearly twice that of other retroviruses. A comparison of the deduced amino acid sequences encoded by the pol region showed HERV-K to be closely related to types A and D retroviruses and even more so to type B retrovirus. It was noted that the env gene product of HERV-K structurally resembles the mouse mammary tumor virus (type B retrovirus) env protein, and the possible expression of the HERV-K env gene in human breast cancer cells is discussed.     

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.     

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.     

Expression of human endogenous retrovirus type K envelope glycoprotein in insect and mammalian cells.

The human endogenous retrovirus type K (HERV-K) family codes for the human teratocarcinoma-derived retrovirus (HTDV) particles. The existence of the envelope protein (ENV) of HERV-K encoded by the subgenomic env mRNA has not yet been demonstrated. To study the genetic requirements for successful expression of ENV, we have constructed a series of recombinant HERV-K env expression vectors for infection and transfection experiments in insect cells and mammalian cells, respectively. Six baculovirus constructs bearing full-length or truncated HERV-K env with or without homologous or heterologous signal peptides were used for infections of insect cells. All recombinant baculoviruses yielded ENV proteins with the expected molecular masses. The full-length 80- to 90-kDa HERV-K ENV protein including the cORF leader sequence was glycosylated in insect cells. In addition, the 14-kDa cORF protein was expressed due to splicing of the full-length env mRNA. The ENV precursor protein is not cleaved to the surface (SU) and transmembrane (TM) glycoproteins; it does not appear on the surface of infected insect cells and is not secreted into the medium. For ENV expression in COS cells, plasmid vectors harboring the cytomegalovirus immediate-early promoter/intron A element and the tissue plasminogen activator (t-PA) signal peptide or the homologous HERV-K signal peptide upstream of the env gene were employed. Glycosylated and uncleaved ENV was expressed as in GH teratocarcinoma cells but at higher levels. The heterologous t-PA signal sequence was instrumental for expression of HERV-K ENV on the cell surface. Hence, we have shown for the first time that the HERV-K env gene has the potential to be expressed as a full-length envelope protein with appropriate glycosylation. In addition, our data provide explanations for the lack of infectivity of HERV-K/HTDV particles.     

Defective retroviruses can disperse in the human genome by intracellular transposition.

Using an assay for retrotransposition detection (T. Heidmann, O. Heidmann, and J. F. Nicolas, Proc. Natl. Acad. Sci. USA 85:2219-2223, 1988), we demonstrated that a defective retrovirus deleted for the gag, pol, and env open reading frames can disperse in the genome of human HeLa cells by intracellular transposition, at a frequency close to 10(-6) events per cell per generation. Transposition requires cooperation in trans for the gag and pol gene products and may be associated with the release of low amounts of noninfectious retroviruslike particles which are the hallmarks but not the intermediates of this transposition process. Similar events could account for the dispersion at high copy number of some of the human endogenous sequences related to retroviruses and for the occurrence of noninfectious retroviruslike particles in human placenta and several tumor cell lines (reviewed by E. Larsson, N. Kato, and M. Cohen, Curr. Top. Microbiol, Immunol, 148:115-132, 1989).     

Proviral amplification of the Gypsy endogenous retrovirus of Drosophila melanogaster involves env-independent invasion of the female germline

Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/restrictive and somatic/germinal lineages. We show here that the derepression of active proviruses in the permissive soma is necessary and sufficient to induce proviral insertions in the progeny, even if the F1 flies derive from restrictive germ cells devoid of active proviruses. Therefore, gypsy endogenous multiplication results from the transfer of some gypsy-encoded genetic material from the soma towards the germen of the mother and its subsequent insertion into the chromosomes of the progeny. This transfer, however, is not likely to result from retroviral infection of the germline. Indeed, we also show here that the insertion of a tagged gypsy element, mutant for the env gene, occurs at high frequency, independently of the production of gypsy Env proteins by any transcomplementing helper. The possible role of the env gene for horizontal transfer to new hosts is discussed.