A placenta-specific receptor for the fusogenic, endogenous retrovirus-derived, human syncytin-2 Ce´ cile Esnaulta,b,1, Ste´ phane Prieta,b,1,2, David Ribeta,b,1,3,Ce´ cile Vernocheta,b, Thomas Brulsc, Christian Laviallea,b, Jean Weissenbachc, and Thierry Heidmanna,b,d,4 aUnite´des Re´trovirus Endoge`nes et Ele´ments Re´troïdes des Eucaryotes Supe´rieurs, Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 8122, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France; bUniversite´Paris-Sud, 91405 Orsay, France; cUnite´deGe´ nomique Me´tabolique, Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 8030, Genoscope, Centre National de Se´quenc¸age, 2 rue Gaston Cre´mieux, 91057 Evry, France; and dPremUP, 4 avenue de l’Observatoire, 75006 Paris, France Edited by John M. Coffin, Tufts University School of Medicine, Boston, MA, and approved September 12, 2008 (received for review July 30, 2008) Syncytin-2 is an envelope gene from the human endogenous retro- surface envelopes, are involved first in cell surface receptor recog- virus FRD (HERV-FRD) co-opted by an ancestral primate host, con- nition and subsequently in viral entry by driving the fusion of the served in evolution over >40 Myr, specifically expressed in the viral envelope with the cell membrane. Expression of Env proteins placenta, and with a cell–cell fusogenic activity likely contributing to at the cell surface can also trigger cell–cell fusion provided that their placenta morphogenesis. Here, using the GeneBridge4 human/Chi- cognate receptors are exposed at the surface of neighboring cells. nese hamster radiation hybrid panel, we mapped and identified the A systematic search through the human genome sequence has human receptor for syncytin-2. This receptor—namely Major Facilita- identified 18 env genes encoding a full-length ORF whose products tor Superfamily Domain Containing 2 (MFSD2)—belongs to a large may potentially have a function (4, 11–13). Among them, the family of presumptive carbohydrate transporters with 10–12 mem- syncytin-1 and -2 proteins are specifically expressed within the brane-spanning domains, is located at chromosomal position 1p34.2, placenta at the cytotrophoblast-syncytiotrophoblast interface and and is conserved in evolution. An expression vector for MFSD2 have been reported to induce cell–cell fusion ex vivo by interacting confers fusogenicity to otherwise insusceptible cells upon trans- with distinct receptors (4, 5, 7, 14, 15). Syncytin-1 was shown to be fection of syncytin-2. It also confers infectivity to syncytin-2 involved in the differentiation and fusion of human cytotropho- pseudotypes, consistent with this protein being the receptor for the blasts in primary cultures (5, 7, 14). In addition, the functionality of ancestrally acquired HERV-FRD family of endogenous retroviruses. At these 2 genes has been conserved in evolution since the time of their variance with the human gene, neither mouse nor rat MFSD2 can insertion into the primate genome (some 25 Myr ago for syncytin-1 mediate membrane fusion, which is consistent with the fact that the and Ͼ40 Myr for syncytin-2), and they currently display remarkably envelope-derived syncytin genes co-opted by rodents during evolu- little polymorphism in the human population (4, 16, 17), 2 strong tion are not orthologous to the human syncytin genes. Remarkably, signs of purifying selection. The host has most probably co-opted a real-time quantitative RT-PCR analysis of MFSD2 in various human these genes of retroviral origin for its own benefit, more specifically, tissues demonstrates specific expression in the placenta, as well as in for syncytiotrophoblast morphogenesis. Understanding the physi- the human BeWo choriocarcinoma cell line, which discloses enhance- ology of syncytiotrophoblast formation and the role of both syn- ment of receptor expression upon induction by forskolin of cell–cell cytins in the fusion process requires identification of their cognate fusion and syncytium formation. In situ hybridization of human receptors and their tissue distribution in the developing placenta. A placental tissue using an MFSD2-specific probe further unambiguously receptor for syncytin-1 has been identified as the RD114/ demonstrates receptor expression at the level of the syncytiotropho- mammalian type D retrovirus receptor, variously referred to as blast, again consistent with a role in placenta morphogenesis. RDR/ASCT2/ATB0/SLC1A5 (5). It is an amino acid transporter envelope protein ͉ human endogenous retrovirus (HERV) ͉ ubiquitously expressed in most human tissues, including the pla- major facilitator superfamily domain containing 2 (MFSD2) ͉ centa, where it is expressed mainly in the cytotrophoblasts (18–20). syncytiotrophoblast Here, we report on the identification of the syncytin-2 receptor via the use of a human/hamster radiation hybrid panel (21, see also 22–24). The identified gene is a receptor with multiple membrane- he placenta is an autonomous and transient organ essentially spanning domains that can mediate cell–cell fusion and infection by aimed at mediating nutrient and gas exchange between mother T retroviral pseudotypes bearing syncytin-2. Remarkably, real-time and fetus during intrauterine life. In several mammalian species RT-PCR on RNA from a large panel of human tissues demon- with a hemochorial placenta—including human—a key process of strates that its expression is placenta-specific and, more precisely, in placental morphogenesis is the fusion of trophoblastic cells into a the syncytiotrophoblast as revealed by in situ hybridization. A multinucleated layer called syncytiotrophoblast, which constitutes model is presented illustrating the primary role of syncytin-2 and its the main materno–fetal barrier in direct contact with maternal newly identified cognate receptor in syncytiotrophoblast formation. blood and fulfils essential trophic exchange functions (1–3). Little is known about the molecular mechanisms involved in trophoblastic differentiation. However, a major advance has been Author contributions: C.E., S.P., D.R., C.V., and T.H. designed research; C.E., S.P., D.R., and made by the identification of envelope (Env) proteins encoded by C.V. performed research; J.W. contributed new reagents/analytic tools; C.E., S.P., D.R., C.V., endogenous retroviruses (ERVs) and likely involved in the forma- T.B., C.L., and T.H. analyzed data; and T.H. wrote the paper. tion of the syncytiotrophoblast (4–8). The mammalian genomes The authors declare no conflict of interest. indeed harbor thousands of ERV elements that display a structure This article is a PNAS Direct Submission. close to that of the integrated proviral form of exogenous retrovi- 1C.E., S.P., and D.R. contributed equally to this work. ruses (gag, pol, and env-related regions flanked by 2 long terminal 2Present address: Architecture et Fonction des Macromole´cules Biologiques, CNRS UMR6098, repeats) and most probably are the remnants of past infections of ESIL case 925, 13288 Marseille Cedex 9, France. the germ line by ancestral retroviruses (9, 10). Although the vast 3Present address: Unite´ des interactions Bacte´ries-Cellules, INSERM U604, INRA USC2020, majority of these elements are defective, a few of them still contain Institut Pasteur, 25 rue du Dr Roux, 75024 Paris Cedex 15, France. intact ORFs, notably in env genes. During the retroviral life cycle, 4To whom correspondence should be addressed. E-mail: [email protected]. Env glycoproteins, which are anchored in the lipid bilayer of viral © 2008 by The National Academy of Sciences of the USA 17532–17537 ͉ PNAS ͉ November 11, 2008 ͉ vol. 105 ͉ no. 45 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807413105 Downloaded by guest on September 30, 2021 A A23 (tk-) HFL121 hamster cells human fibroblasts LacZ viral RNA lethal irradiation SIV viral core syn2 fusion (3,000 rads) infection with selection transfection SIV-syn2 seeding pseudotype into plate 293T panel of 93 hybrids X-gal staining and producer cells GeneBridge4 determination of viral titer (LacZ+ cfu) B 180 160 140 Fig. 1. Rationale of the retroviral pseudotype assay 120 cfu/ml) for the screening of the GeneBridge4 human/ + 100 Chinese hamster Radiation Hybrid panel and map- ping of the human syncytin-2 receptor MFSD2. (A) 80 The 93 hybrid clones of the GeneBridge4 panel (21) 60 generated as schematized (Left) were seeded and 40 infected with viral pseudotypes produced by co- viral titer (LacZ transfection of human 293T cells with expression 20 vectors for the SIV core, the syncytin-2 Env protein, 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 A and a lacZ-containing retroviral transcript (Right). hybrid clone number Viral titers were determined 3 days after infection by X-gal staining of the cells. (B) Pseudotype viral titers 1p34.2 obtained in a representative experiment involving C the simultaneous assay of the 93 clones of the panel Chr.1 p31.1 q12 q41 (and control A23 cells, ‘‘A’’ at the right end of the AFMB338WG5 abscissa axis), with each cell clone infected in dupli- cate and examined for the number of lacZϩ foci. The resulting matrix was derived and data were analyzed AFMB338WG5 39.3 40.3 by using the RH map program. (C) Localization of the identified genetic marker (AFMb3385) on chromo- MACF1 HEYL PPIE TRIT1 MFSD2 CAP1 some 1, with position of the genes present within 1 BMP8A NT5C1A BMP8B MYCL1 Mb, as determined by using the UCSC Genome 0.1 Mb PABPC4 HPCAL4 Browser. The MFSD2 gene is arrowed. Results taining 2 (MFSD2), and therefore was a good candidate as a Identification of the Syncytin-2 Receptor. We had shown that syn- retrovirus receptor (reviewed in refs. 28 and 29). A complete cDNA cytin-2, as a bona fide retroviral Env protein, can be incorporated clone was obtained from Invitrogen and introduced into a CMV- into env-defective lentiviral virions [e.g., simian immunodeficiency driven expression vector.