Guy Oriol

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

ABSTRACT 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.

Synthesis, Assembly, and Processing of the Env ERVWE1/Syncytin Human Endogenous Retroviral Envelope

ABSTRACT 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.

A Retroviral Promoter and a Cellular Enhancer Define a Bipartite Element Which Controls env ERVWE1 Placental Expression

ABSTRACT 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.

Identification of the hASCT2-binding domain of the Env ERVWE1/syncytin-1 fusogenic glycoprotein

The cellular HERV-W envelope/syncytin-1 protein, encoded by the envelope gene of the ERVWE1 proviral locus is a fusogenic glycoprotein probably involved in the formation of the placental syncytiotrophoblast layer. Syncytin-1-induced in vitro cell-cell fusion is dependent on the interaction with hASCT2. As no receptor binding domain has been clearly defined in the SU of neither the HERV-W Env nor the retroviruses of the same interference group, we designed an in vitro binding assay to evaluate the interaction of the HERV-W envelope with the hASCT2 receptor. Using truncated HERV-W SU subunits, a region consisting of the N-terminal 124 amino acids of the mature SU glycoprotein was determined as the minimal receptor-binding domain. This domain contains several sub-domains which are poorly conserved among retroviruses of this interference group but a region of 18 residus containing the SDGGGX2DX2R conserved motif was proved to be essential for syncytin-1-hASCT2 interaction.

Natural history of the ERVWE1 endogenous retroviral locus

BackgroundThe human HERV-W multicopy family includes a unique proviral locus, termed ERVWE1, whose full-length envelope ORF was preserved through evolution by the action of a selective pressure. The encoded Env protein (Syncytin) is involved in hominoid placental physiology.ResultsIn order to infer the natural history of this domestication process, a comparative genomic analysis of the human 7q21.2 syntenic regions in eutherians was performed. In primates, this region was progressively colonized by LTR-elements, leading to two different evolutionary pathways in Cercopithecidae and Hominidae, a genetic drift versus a domestication, respectively.ConclusionThe preservation in Hominoids of a genomic structure consisting in the juxtaposition of a retrotransposon-derived MaLR LTR and the ERVWE1 provirus suggests a functional link between both elements.

CONSTRUCTION AND EXPRESSION OF A MODULAR GENE ENCODING BACTERIOPHAGE T7 RNA POLYMERASE

A modular gene that encodes T7 RNA polymerase (T7 RNAP) and consists of cassettes delimited by unique restriction sites was constructed. The modular and wild-type genes of T7 RNAP were cloned into a vector designed to express His-tagged proteins. The modular and wild-type genes provided the same level of protein expression (i.e., T7 RNAP represented up to 30% of the total protein in Escherichia coli strain BL21). Purification of both proteins by immobilized metal ion affinity chromatography (IMAC) resulted in similar yields (700-800 microg of enzyme per 20 ml of culture) and purity (>95%) as indicated by Coomassie blue staining, Western blotting and the absence of detectable contaminating nuclease activities. Both proteins exhibited identical efficiency in transcription assays, and their specific activities (about 200 U/microg) were close to that of a commercial T7 RNAP preparation. The modular gene provides a useful tool for cassette directed mutagenesis of T7 RNAP.

Molecular characterization of HIV viruses generated after in vivo ligation

During the course of infection, human immunodeficiency virus type 1 (HIV-1) displays wide genotypic and phenotypic differences. Construction of chimeric viruses is useful to determine the genotypic basis that underlies phenotypic variations, but the procedure is time-consuming. Previously, it has been shown that co-transfection of truncated hemi-genomic HIV-1 proviral DNA can lead to generation of full-length infectious virus. In the study of HIV phenotypes, using this technique, it is important to determine whether recombination between the two hemigenomes occurs without mutations. After co-transfection, progeny recombinant viruses replicated at the same rate as the control. We purified progeny viruses from culture supernatants and determined mutations at the recombination site. It appeared that correct in vivo ligation depended on the purity of DNA and the restriction site used. It also appeared that some of the mutations observed affect replication, as progeny viruses bearing one of these mutations disappeared during in vitro cultures, whereas other mutants did not. Although this technique is widely applied to generate chimeric viruses, the results should be evaluated with care, since mutations influencing the phenotype of the progeny viruses may have been introduced.