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Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new "syncytin" in a third order of mammals - PubMed

  • ️Thu Jan 01 2009

Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new "syncytin" in a third order of mammals

Odile Heidmann et al. Retrovirology. 2009.

Abstract

Background: Syncytins are envelope genes of retroviral origin that have been co-opted by the host to mediate a specialized function in placentation. Two of these genes have already been identified in primates, as well as two distinct, non orthologous genes in rodents.

Results: Here we identified within the rabbit Oryctolagus cuniculus-which belongs to the lagomorpha order- an envelope (env) gene of retroviral origin with the characteristic features of a bona fide syncytin, that we named syncytin-Ory1. An in silico search for full-length env genes with an uninterrupted open reading frame within the rabbit genome first identified two candidate genes that were tested for their specific expression in the placenta by quantitative RT-PCR of RNA isolated from a large set of tissues. This resulted in the identification of an env gene with placenta-specific expression and belonging to a family of endogenous retroelements present at a limited copy number in the rabbit genome. Functional characterization of the identified placenta-expressed env gene after cloning in a CMV-driven expression vector and transient transfection experiments, demonstrated both fusogenic activity in an ex vivo cell-cell fusion assay and infectivity of pseudotypes. The receptor for the rabbit syncytin-Ory1 was found to be the same as that for human syncytin-1, i.e. the previously identified ASCT2 transporter. This was demonstrated by a co-culture fusion assay between hamster A23 cells transduced with an expression vector for ASCT2 and A23 cells transduced with syncytin-Ory1. Finally, in situ hybridization of rabbit placenta sections with a syncytin-Ory1 probe revealed specific expression at the level of the junctional zone between the placental lobe and the maternal decidua, where the invading syncytial fetal tissue contacts the maternal decidua to form the labyrinth, consistent with a role in the formation of the syncytiotrophoblast. The syncytin-Ory1 gene is found in Leporidae but not in Ochotonidae, and should therefore have entered the lagomorpha order 12-30 million years ago.

Conclusion: The identification of a novel syncytin gene within a third order of mammals displaying syncytiotrophoblast formation during placentation strongly supports the notion that on several occasions retroviral infections have resulted in the independent capture of genes that have been positively selected for a convergent physiological role.

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Figures

Figure 1
Figure 1

Retroviral envelope protein-based phylogenic tree with positions of the identified rabbit Env-Ory1- and Env-Ory2, and of the human and mouse syncytins. The tree was determined by the neighbor-joining method using envelope TM subunit sequences (see ref [12]) from murine and human ERVs, and infectious retroviruses. The horizontal branch length and the scale indicate the percentage of amino acid substitutions from the node. Percent bootstrap values obtained from 1,000 replicates are indicated.

Figure 2
Figure 2

Real-time quantitative RT-PCR analysis of Env-Ory1 and Env-Ory2 transcripts in rabbit tissues. Transcript levels were normalized relative to the amount of 18S rRNA (arbitrary units). At least 3 samples per organ type were analyzed for Env-Ory1 (from different adult animals for non-fetal tissues; from a given litter for the embryos and placentae); one sample per organ type was analyzed for Env-Ory2.

Figure 3
Figure 3

Characterization of Env-Ory1. (A) Schematic representation of the Env-Ory1-associated ERV with the LTRs and the splice sites for the sub-genomic env transcript indicated. (B) Schematic structure, hydrophobicity profile and primary sequence of the Env-Ory1 glycoprotein (deposited in GenBank [GenBank:GU196371]). The SU and TM subunits of the envelope protein are delineated, with a canonical furin cleavage site (RQKR; consensus: R/K-N-R/K-R) between the two subunits and the CWLC domain involved in SU-TM interaction indicated in red; the hydrophobic signal peptide and fusion peptide and the transmembrane domain are shaded in light gray, and the putative immunosuppressive domain (ISU) in dark gray.

Figure 4
Figure 4

Fusogenic activity of syncytin-Ory1. (A) Assay for cell-cell fusion mediated by syncytin-Ory1. The indicated cell lines were transfected with an expression vector for syncytin-Ory1 or an empty vector (none) together with a LacZ expression vector. Cells were cultured for 1-2 days after transfection, fixed and X-gal-stained. Syncytia (arrows) were detected in syncytin-Ory1-transfected SH-SY5Y cells, with only mononucleated cells visible in the other cases. (B) Assay for cell infection mediated by syncytin-Ory1-pseudotyped virus particles. Pseudotypes were produced by cotransfection of human 293T cells with expression vectors for the SIV core, the syncytin-Ory1 protein (or an empty vector) and a LacZ-containing retroviral transcript. Supernatants were used to infect the indicated target cells, which were X-gal stained 3 days after infection. Abbreviation: Syn-Ory1, syncytin-Ory1.

Figure 5
Figure 5

Fusion assay between ASCT2-transduced and syncytin-Ory1-transduced co-cultured cells demonstrates that ASCT2 is the syncytin-Ory1 receptor. Left panel: Cell-cell fusion was assayed upon independent transfections of a set of A23 cells with an empty vector (none) or an expression vector for either the syncytin-Ory1, syncytin-1 or syncytin-2 protein together with an nls-LacZ gene-expression vector, and another set of A23 cells with an expression vector for the syncytin-1 receptor ASCT2, the syncytin-2 receptor MFSD2 [13] or an empty vector (none). One day after transfection, cells were resuspended and pairs of transfected cells from each set were cocultured for 1-2 days, fixed and X-Gal stained. Right panel: Syncytia can be easily detected (arrows) for the syncytin-Ory1/ASCT2, syncytin-1/ASCT2 and syncytin-2/MFSD2 pairs, with only mononucleated cells visible in the other cases. Abbreviations: syn-Ory1, syncytin-Ory1; syn1, syncytin-1; syn2, syncytin-2.

Figure 6
Figure 6

Structure and in situ hybridization for syncytin-Ory1 expression of day 12 rabbit placenta: (A) Schematic representation of a rabbit placenta (right) and haematoxylin and eosin staining of a day 12 placenta section (left) with the 3 main layers of the placenta indicated. (B) Higher magnification of the areas framed in A. Abbreviations: frbc: fetal red blood cell, fv: fetal blood vessel, ml: maternal blood lacuna, mrbc: maternal red blood cell. (C) In situ hybridization on sections of a day 12 rabbit placenta (serial sections of the HES in B) with digoxigenin-labeled syncytin-Ory1 sense (lower panel, negative control) and antisense (upper panel) riboprobes, revealed with an alkaline phosphatase-conjugated anti-digoxigenin antibody. Brackets and arrows highlight the positive labeling of trophoblast cells surrounding the invading fetal vessels in the junctional zone.

Figure 7
Figure 7

Putative entry date of syncytin-Ory1 during lagomorph evolution. Schematized phylogenetic tree with the evolutionary timeline of four lagomorph genus (adapted from [16]) and the rodent and primate outgroups depicted, with average divergence times indicated for the nodes. The presence of syncytin-Ory1 sequences in each genus, detected either by PCR experiments (a) or database screening (b), is indicated on the right.

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