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. 2003 Oct 28;100(22):13013-8.
doi: 10.1073/pnas.2132646100. Epub 2003 Oct 13.

Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution

Sandra Blaise  1 , Nathalie de Parseval, Laurence Bénit, Thierry Heidmann
Affiliations

Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution

Sandra Blaise et al. Proc Natl Acad Sci U S A. .

Abstract

Screening human sequence databases for endogenous retroviral elements with coding envelope genes has revealed 16 candidate genes that we assayed for their fusogenic properties. All 16 genes were cloned in a eukaryotic expression vector and assayed for cell-cell fusion by using a large panel of mammalian cells in transient transfection assays. Fusion was observed for two human endogenous retrovirus (HERV) envelopes, the previously characterized HERV-W envelope, also called syncytin, and a previously uncharacterized gene from the HERV-FRD family. Cells prone to env-mediated fusion were different for the two envelopes, indicating different receptor usage. A search for the FRDenv gene in primates indicated that the corresponding proviral element is present in all simians, from New World monkeys to humans, being absent only in prosimians. Cloning of the corresponding env genes in simians disclosed conservation of the fully coding status of the gene, and most remarkably, conservation of its fusogenic property. Finally, a Northern blot analysis for the expression of the FRD family among a series of human tissues demonstrated specific expression in the placenta, as previously demonstrated for the other fusogenic human envelope of the HERV-W family. Altogether, the present data have identified a previously uncharacterized envelope (that we propose to name syncytin 2 after renaming syncytin as syncytin 1) with a potential role in placenta formation, and the identification of the complete set of retroviral envelopes with fusogenic properties now allows a definite analysis of the possible role of HERV in this physiological process, via classical genetic approaches.

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Figures

Fig. 4.
Fig. 4.
Conservation of the HERV-FRD env locus and ORF on primate evolution. (A) Slot-blot of genomic DNA from simian, prosimian, cat, and mouse species, probed with a full-length HERV-FRD env fragment. The inferred date of insertion of HERV-FRD is indicated with an arrow on the primate phylogenetic tree on the left. NWM, New World monkey. (B) In vitro transcription/ translation assay of the human HERV-FRD env gene and of the orthologous genes from the indicated simian species. Env genes were PCR-amplified as schematized, submitted to in vitro transcription/translation (“control” is without DNA template), run on a polyacrylamide gel, and autoradiographed. The arrow points to the bands of the expected size.
Fig. 1.
Fig. 1.
Envelope-mediated cell–cell fusion. (A) Construction of envelope-expressing vectors and rationale of the fusion assay. Each of the 16 envelope genes was PCR-amplified from BAC DNA and cloned into the phCMV expression vector. The env genes inserted in between the β-globin intron and pA sequences are schematized with the putative cleavage site between the SU and the TM envelope subdomains. Cells were transfected with the env-expressing vectors and stained with May–Grünwald and Giemsa solutions 12 h (for the G355-5 cells) or 36 h (for the other cell types) after transfection. Fusion indices were calculated as indicated in Materials and Methods. (B) Syncytia formation by HERV envelope glycoproteins in various cell types. Cells were transfected with vectors expressing the HERV-FRD envelope (envFRD), the HERV-W envelope (envW), or an empty vector (none).
Fig. 2.
Fig. 2.
Primary sequence, hydrophobicity profile, and predicted features of the HERV-FRD envelope. The SU and TM moieties of the envelope are delineated, with the canonical RVRR cleavage site between the two subunits underlined (consensus: R/K-X-R/K-R); in the TM subunit, the hydrophobic fusion peptide and transmembrane domains are shaded in light gray, and the putative immunosuppressive domain (ISU) in dark gray; in the SU subunit, the canonical CWLC domain involved in SU-TM interaction is underlined.
Fig. 3.
Fig. 3.
Northern blot analysis of HERV-FRD env expression in human tissues. Poly(A)+ RNAs (Ambion, Human Northern Blot 2) were probed with an antisense-strand HERV-FRD env riboprobe. Exposure time was 12 h.
Fig. 5.
Fig. 5.
Fusion activity of the orthologous primate ERV-FRD envelope glycoproteins. The indicated target cells were transfected as indicated in Fig. 1 with vectors expressing the human or simian orthologous ERV-FRD envelopes, and fusion indices were determined as indicated in Table 2 (means ± SDs; n = 5).
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