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. 2013 Feb 26;110(9):E828-37.
doi: 10.1073/pnas.1215787110. Epub 2013 Feb 11.

Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants

Guillaume Cornelis  1 , Odile Heidmann, Séverine A Degrelle, Cécile Vernochet, Christian Lavialle, Claire Letzelter, Sibylle Bernard-Stoecklin, Alexandre Hassanin, Baptiste Mulot, Michel Guillomot, Isabelle Hue, Thierry Heidmann, Anne Dupressoir
Affiliations

Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants

Guillaume Cornelis et al. Proc Natl Acad Sci U S A. .

Abstract

Syncytins are envelope genes of retroviral origin that have been co-opted for a role in placentation and likely contribute to the remarkable diversity of placental structures. Independent capture events have been identified in primates, rodents, lagomorphs, and carnivores, where they are involved in the formation of a syncytium layer at the fetomaternal interface via trophoblast cell-cell fusion. We searched for similar genes within the suborder Ruminantia where the placenta lacks an extended syncytium layer but displays a heterologous cell-fusion process unique among eutherian mammals. An in silico search for intact envelope genes within the Bos taurus genome identified 18 candidates belonging to five endogenous retrovirus families, with one gene displaying both placenta-specific expression, as assessed by quantitative RT-PCR analyses of a large panel of tissues, and conservation in the Ovis aries genome. Both the bovine and ovine orthologs displayed fusogenic activity by conferring infectivity on retroviral pseudotypes and triggering cell-cell fusion. In situ hybridization of placenta sections revealed specific expression in the trophoblast binucleate cells, consistent with a role in the formation--by heterologous cell fusion with uterine cells--of the trinucleate cells of the cow and the syncytial plaques of the ewe. Finally, we show that this gene, which we named "Syncytin-Rum1," is conserved among 16 representatives of higher ruminants, with evidence for purifying selection and conservation of its fusogenic properties, over 30 millions years of evolution. These data argue for syncytins being a major driving force in the emergence and diversity of the placenta.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Multiple Syncytin captures and diversity of placental structures in eutherian mammals. (Left) The phylogeny of eutherians that can be grouped into four major clades: (I) Afrotheria, (II) Xenarthra, (III) Euarchontoglires, and (IV) Laurasiatheria (adapted from ref. 46). The different types of placentation are indicated by colored squares (the color code is given in the scheme to the right). The time of insertion of the different syncytin genes is shown. Branch length is proportional to time (in million years, My). (Right) Schematic color-coded representation of the fetomaternal interface in the four main types of placental structures. Placental types are classified from top to bottom in the order of decreasing invasive properties and extent of syncytialization. The synepitheliochorial placentation of Ruminantia is unique among eutherians and is characterized by a heterologous cell-fusion process between cells of fetal and maternal origin and a very limited extent of syncytialization.
Fig. 2.
Fig. 2.
Structure of a canonical retroviral Env protein and characterization of the identified bovine candidates. (A) Schematic representation of a retroviral Env protein, delineating the SU and TM subunits. The furin cleavage site (consensus: R/K-X-R/K-R) between the two subunits, the C-X-X-C motif involved in SU–TM interaction, the hydrophobic signal peptide (purple), the fusion peptide (green), the transmembrane domain (red), the putative ISD (blue), and the conserved C-X5/6/7-C motif are indicated. (B) Characterization of the candidate bovine Env proteins. (Left) The hydrophobicity profile for each candidate is shown with the position of the canonical structural features highlighted in A shown when present. The color code is as in A. (Right) number of full-length env gene ORFs within each family of elements. The total number of genomic copies is shown in parentheses.
Fig. 3.
Fig. 3.
Retroviral Env protein-based phylogenetic tree with the identified Bos-Env protein candidates. The maximum-likelihood tree inferred with the RAxML software was constructed using Env protein amino acid sequences from murine and human ERVs and from a series of infectious retroviruses. The horizontal branch length is proportional to the percentage of amino acid substitutions from the node (the scale bar is shown on the left), and the percent bootstrap values obtained from 1,000 replicates are indicated at the nodes. The clusters of 13 and 2 Env proteins that were grouped into single families of elements (Bos-Env2 and Bos-Env5, respectively) are distinguished by indication of their chromosomal position (Table S2). Bos-Env4 and Bos-Env5 (chr24) correspond to the Env of the previously identified bovine ERVs BERV-K1 and -K2 (23, 24), respectively. BFV, bovine foamy virus; BLV, bovine leukemia virus; ENTV, enzootic tumorigenic virus; FeLV, feline leukemia virus; JSRV, Jaagsiekte retrovirus; HERV, human ERV; HFV, human foamy virus; HIV, human immunodeficiency virus; MoMLV, Moloney murine leukemia virus; mIAPE, Mus musculus intracisternal A-type particle with an envelope gene; MMTV, murine mammary tumor virus; MPMV, Mason–Pfizer monkey virus; OMVV, ovine Maedi-Visna virus; PERV, porcine ERV.
Fig. 4.
Fig. 4.
qRT-PCR analysis of the candidate env gene transcripts from the cow. Transcript levels are expressed as the ratio of the expression level of each env gene to that of the SDHA control gene (Methods). Note the enlarged vertical scale for the two placenta-specific bos-env1 and bos-env4 genes. Fetal villous tissue at d62 was used as the bovine placental sample, and the corresponding interplacentomal uterine endometrium from the mother was analyzed in parallel. The results obtained for the five env gene candidates in the same series of tissues are shown (tissues are displayed in the same order in all panels; tissue names are abbreviated in the upper right and three lower panels). Values for the placenta are the means of at least three samples; error bars show the mean ± SEM.
Fig. 5.
Fig. 5.
Characterization of the bos-env1–, ovis-env1–, and bos-env4–containing ERVs and of their genomic location in the cow and sheep. (A) Characterization of the bos-env1–containing ERV. (Upper) Structure of the bos-env1–containing ERV and evidence for orthology between the cow and sheep env1 sequences. Homologous regions common to both sequences are aligned. Repeated mobile elements (gray) as identified by the RepeatMasker web program are positioned. Of note, bos-env1 is in a provirus directly followed by a tandem repeat of a homologous provirus sharing a common LTR. The proviral LTRs, the degenerate gag-pol and env genes, and the env1 gene ORF sequence are indicated (the key for symbols used is shown below the panel). PCR primers used to identify the bos-env1 orthologous copy in the sheep (black half arrows) and splice sites for the env subgenomic transcripts as determined by RT-PCR of cow placental RNA or by alignment with an EST sequence for the sheep are indicated. The nonhomologous sequence in the sheep LTR positioned 3′ to the env1 sequence compared with the cow corresponds to a 200-bp direct tandem duplication of part of the LTR 3′ end in the sheep. (Lower) Absence of the bos-env1–containing ERV in the genomes of distant mammalian lineages. The bos-env1–containing provirus (in yellow, with its LTRs schematized by boxed triangles and the env gene in red) was used as a reference, and synteny between the cow, pig, dog, mouse, and human genomes was determined with the Comparative Genomic tool of the UCSC Genome Browser (http://genome.ucsc.edu/). The positions of exons (vertical lines) of the resident B4GALT5 and PTGIS gene and the sense of transcription (arrows) are indicated. Homologous regions are shown as black boxes, nonhomologous regions as thin lines (not to scale), and gaps as dotted lines. (B) Structure of the bos-env4–containing ERV and evidence of the absence of proviral integration in the sheep syntenic locus. The symbol code is as in A. Homologous sequences flanking the provirus in the cow and colinear in the sheep are indicated by bold lines. Comparison of the flanking sequences of the cow provirus with those of the empty locus in the sheep (obtained by PCR with the indicated primers) provides evidence for target-site duplication in the cow (red boxes), a characteristic feature of retroviral integration.
Fig. 6.
Fig. 6.
Bos- and Ovis-Env1 are fusogenic retroviral Env proteins. (A) Bos-Env1 and Ovis-Env1 are fusogenic in pseudotyping assays. (Upper) Schematic representation of the assay for cell infection with Bos-Env1– or Ovis-Env1–pseudotyped virus particles. Pseudotypes are produced by cotransfection of human 293T cells with expression vectors for the MLV core, the Bos-Env1 or Ovis-Env1 proteins (or either ecotropic-MLV Env or an empty vector as controls), and a plasmid expressing a nuclear β-galactosidase encoded by a nlsLacZ-containing retroviral transcript. Pseudotyped virus particles in cell supernatants then are assayed for infection of the indicated target cells, which are stained with X-Gal (3-d postexposure). (Lower Left) X-Gal–stained target cells exposed to particles pseudotyped with Bos-Env1, Ovis-Env1, or, as a negative control, Env from an ecotropic MLV (infecting only murine cells) on cow MDBK target cells. (Lower Right) As a positive control, the viral titer of particles pseudotyped with ecotropic MLV-Env was tested on murine WOP cells. (B) Viral titers for particles pseudotyped with Bos-Env1 or Ovis-Env1 assayed on a panel of target cells from cow (MDBK), pig (PK), carnivores [cat (G355.5) or dog (A72)], human (HuH7, 293T, and SH-SY5Y), or rodents [mouse (WOP and LOK) or hamster (A23)]. Titers, expressed as focus-forming units (ffu) per milliliter ± SD, are corrected for the background values of control particles without an Env protein and are the means from at least three independent experiments. (C) Bos-Env1 and Ovis-Env1 are fusogenic in a cell–cell fusion assay. (Upper) Schematic representation of the cell–cell fusion assay with cells cotransfected with a plasmid expressing Bos-Env1 or Ovis-Env1 (or an ecotropic-MLV Env, a C-terminal–truncated amphotropic-MLV Env, or an empty vector as controls) and a plasmid expressing a nuclear β-galactosidase (nlsLacZ). Twenty-four hours after transfection, cells were treated transiently (5 min) with neutral or acidic PBS buffer (pH 7 or pH 5) and were stained with X-Gal 4–6 h posttreatment. (Lower) G355.5 cat cells were transfected with Bos-Env1, Ovis-Env1, or, as a negative control, Env from an ecotropic MLV or an empty vector, and neutral (Upper Row) or acidic (Lower Row) pH treated. As a positive control, cells were transfected with the C-terminal–truncated (R-less) amphotropic-MLV Env (Amphotropic-MLV Env*) (Right) (30).
Fig. 7.
Fig. 7.
Structure of the synepitheliochorial placenta of higher ruminants and in situ hybridization for syncytin-Rum1 expression on cow and sheep placental sections. (A) (Left) Schematic drawing of the bovine fetus and placenta in utero. The yellow and gray areas represent the fetus and mother membranes, respectively; the localized areas of formation of the fetomaternal villous units, the placentomes (pl), are indicated. The maternal (red) and fetal (blue) vessels are schematized. (Right) Detailed scheme of a bovine placentome. The placental fetal villi are intimately enmeshed with preformed maternal endometrial crypts (both covered by their respective epithelium) for maximal exchanges between the fetal and maternal blood circulations. Of note, the placentome organization in the cow and sheep is identical, except that on the fetal side it is convex in the cow and concave in the sheep. (B) Diagram of the synepitheliochorial fetomaternal barrier of ruminants. Binucleate cells (BNCs) residing in the trophoblast epithelium and possessing characteristic granules migrate into the uterine epithelium and fuse with the apex of a single uterine epithelial cell forming a fetomaternal hybrid trinucleate cell. BNC granules then are released on the maternal side of the placenta. In the sheep, the fusion process involving the BNCs proceeds further and generates multinucleate syncytial plaques which partially replace the uterine epithelium. (C and D) ISH on serial sections of placenta from cow and sheep, respectively, at the first trimester, observed at different magnifications, using digoxigenin-labeled antisense or sense riboprobes revealed with an alkaline phosphatase-conjugated anti-digoxigenin antibody. (C) Partial view of a placentome (pl) and endometrium (en) zona, with the fetal villi (fvi), the maternal crypts (mc), the uterine stroma (st), and the labeled BNCs (arrowheads) indicated. (D) Higher magnification of a fetomaternal interhemal area (see scheme in B); the maternal crypts and fetal villi, the labeled BNCs, the fetal and maternal vessels, the uterine stroma, and the position of the uterine epithelium in the cow or of the syncytial plaque in the sheep are schematized to the right of each panel. Note that the syncytial plaque, the uterine epithelium, and the stroma are not labeled. (Scale bars: 200 µm in C and 25 µm in D.)
Fig. 8.
Fig. 8.
Relative expression level of the syncytin-Rum1 gene in the bovine and ovine placenta during gestation. The mRNA levels were quantified by qRT-PCR using a unique pair of primers designed from regions that are identical in the bovine and ovine syncytin-Rum1. Transcript levels are expressed relative to the lowest value taken as unity and were normalized relative to the amount of the gene encoding succinate dehydrogenase, subunit A (SDHA) (logarithmic scale). Placenta tissues were obtained at the indicated days of gestation (comparable gestation stages are grouped). Vertical arrows indicate two major quantitative changes in the BNC fusion processes occurring during pregnancy (16, 17). Note the significant 3.5- to 10-fold increase in both the bovine and ovine syncytin-Rum1 transcript levels observed at the initiation of the BNC fusion processes and the strong six- to sevenfold increase in the ovine (but not bovine) syncytin-Rum1 transcript level concomitant with the increase in size of the syncytial plaques in sheep placenta. The higher level of syncytin-Rum1 expression could not be explained by a higher proportion of BNCs in the sheep than in the cow, because the percentage of BNCs in the trophectoderm (around 20%) is almost identical in both species throughout the gestation period (31). dpc, days post coitum.
Fig. 9.
Fig. 9.
Status of syncytin-Rum1 during the radiation of the Ruminantia. The Ruminantia phylogenetic tree is shown with the two infraorders, Pecora (higher ruminants; most of the ruminant families) and Tragulina (only one extant family), as well as the outgroups Cetacea, Suina, and Tylopoda (adapted from refs. and 48). The phylogeny of Pecora is not fully resolved (44, 45). Branches whose interrelationship is still under question are represented as paraphyletic. The names of the 17 Ruminantia species tested for the presence of the syncytin-Rum1 gene are given, together with the length (in amino acids) of the Syncytin-Rum1 proteins that were identified for each species (all sequences were deposited in GenBank, accession numbers JX412964–JX412985); the fusogenic activity for each cloned gene, as determined by the pseudotyping assay in Fig. 6 C and D, is provided. Asterisks indicate syncytin-Rum1 genes PCR-amplified with their 3′ flanking sequence; a indicates PCR-amplification of more than one syncytin-Rum1 full-length ORF. For Antilocapra americana, one gene (GenBank accession number JX412981) among the five tested was found to be fusogenic. n.d., not demonstrated; n.r., not relevant; ±, only partial syncytin-Rum1 sequence was obtained.
Fig. 10.
Fig. 10.
Evidence for sequence conservation of and selection pressure on the syncytin-Rum1 gene. (A, Left) Syncytin-Rum1–based maximum-likelihood phylogenetic tree determined using amino acid alignment of the Syncytin-Rum1 proteins identified in Fig. 9, inferred with the RAxML program. The horizontal branch length and scale indicate the percentage of amino acid substitutions. Percent bootstrap values obtained from 1,000 replicates are indicated at the nodes. An asterisk indicates that the sequence used for Axis axis is a consensus of the four full-length gene ORFs (otherwise branching together) and, for Antilocapra americana, the one shown to be fusogenic in the pseudotyping assay (Fig. 9). (A, Right) Double-entry table for the pairwise percentage of amino acid sequence identity between the syncytin-Rum1 genes belonging to the indicated species. (B) Site-specific analysis of selection on syncytin-Rum1 gene codons, using the PAML (M8 model) package. The relevant selection indexes are provided for each codon. A schematic representation of the Syncytin-Rum1 protein domains is given also; conventions are as in Fig. 2. Significant values (P ≥ 0.95) are represented as red dots.
Fig. P1.
Fig. P1.
A syncytin gene associated with the synepitheliochorial placenta of Ruminantia. (Left) Phylogeny of eutherian mammals [grouped into four major clades: (I) Afrotheria, (II) Xenarthra, (III) Euarchontoglires, and (IV) Laurasiatheria], with the different types of placentation indicated by colored squares according to the color code at the right]. The time of insertion of the previously identified syncytin genes (purple triangles) is shown also. (Right) Maternal–fetal interface in the four main types of placental structures. The synepitheliochorial placentation of Ruminantia is unique among eutherians and is characterized by heterologous cell fusion between cells of fetal and maternal origin and a very limited extent of syncytialization.
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