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. 2014 Jul;88(14):7915-28.
doi: 10.1128/JVI.00141-14. Epub 2014 Apr 30.

Capture of syncytin-Mar1, a fusogenic endogenous retroviral envelope gene involved in placentation in the Rodentia squirrel-related clade

François Redelsperger  1 , Guillaume Cornelis  2 , Cécile Vernochet  1 , Bud C Tennant  3 , François Catzeflis  4 , Baptiste Mulot  5 , Odile Heidmann  1 , Thierry Heidmann  6 , Anne Dupressoir  1
Affiliations

Capture of syncytin-Mar1, a fusogenic endogenous retroviral envelope gene involved in placentation in the Rodentia squirrel-related clade

François Redelsperger et al. J Virol. 2014 Jul.

Abstract

Syncytin genes are fusogenic envelope protein (env) genes of retroviral origin that have been captured for a function in placentation. Within rodents, two such genes have previously been identified in the mouse-related clade, allowing a demonstration of their essential role via knockout mice. Here, we searched for similar genes in a second major clade of the Rodentia order, the squirrel-related clade, taking advantage of the complete sequencing of the ground squirrel Ictidomys tridecemlineatus genome. In silico search for env genes with full coding capacity identified several candidate genes with one displaying placenta-specific expression, as revealed by quantitative reverse transcription-PCR analysis of a large panel of tissues. This gene belongs to a degenerate endogenous retroviral element, with recognizable hallmarks of an integrated provirus. Cloning of the gene in an expression vector for ex vivo cell-cell fusion and pseudotype assays demonstrated fusogenicity on a large panel of mammalian cells. In situ hybridization on placenta sections showed specific expression in domains where trophoblast cells fuse into a syncytiotrophoblast at the fetomaternal interface, consistent with a role in syncytium formation. Finally, we show that the gene is conserved among the tribe Marmotini, thus dating its capture back to about at least 25 million years ago, with evidence for purifying selection and conservation of fusogenic activity. This gene that we named syncytin-Mar1 is distinct from all seven Syncytin genes identified to date in eutherian mammals and is likely to be a major effector of placentation in its related clade. Importance: Syncytin genes are fusogenic envelope genes of retroviral origin, ancestrally captured for a function in placentation. Within rodents, two such genes had been previously identified in the mouse-related clade. Here, in the squirrel-related rodent clade, we identified the envelope gene of an endogenous retrovirus with all the features of a Syncytin: it is specifically expressed in the placenta of the woodchuck Marmota monax, at the level of cells fusing into a syncytium; it can trigger cell-cell and virus-cell fusion ex vivo; and it has been conserved for >25 million years of evolution, suggesting an essential role in its host physiology. Remarkably, syncytin-Mar1 is unrelated to all other Syncytin genes identified thus far in mammals (primates, muroids, carnivores, and ruminants). These results extend the range of retroviral envelope gene "domestication" in mammals and show that these events occurred independently, on multiple occasions during evolution to improve placental development in a process of convergent evolution.

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Figures

FIG 1
FIG 1
Phylogeny of Euarchontoglires and previously identified Syncytin genes. Euarchontoglires can be grouped into three major orders: Rodentia, Lagomorpha, and Primates (dates are from reference 36). Rodentia can be further subdivided into three major clades: the squirrel-related clade, the mouse-related clade and the guinea-pig-related clade Ctenohystrica (37). Branch length is proportional to time (in million years [My]), and the families where syncytins have been identified to date are indicated with the corresponding gene name.
FIG 2
FIG 2
Structure of a canonical retroviral envelope protein and characterization of the identified Ictidomys tridecemlineatus candidates. (A) Schematic representation of a retroviral envelope protein, with the surface (SU) and transmembrane (TM) subunits delineated, and the furin cleavage site (consensus, R/K-X-R/K-R) between the two subunits together with the C-X-X-C domain involved in SU-TM interaction indicated; the hydrophobic signal peptide (purple), fusion peptide (green), transmembrane domain (red), and putative immunosuppressive domain (ISD) (blue) are also indicated. (B) Characterization of the thirteen-lined ground squirrel candidate envelope proteins. (Left) The hydrophobicity profile for each candidate is shown with the positioning of the canonical structural features schematized in panel A, when present (same color code). (Right) Number of full-length env gene ORFs within each family of element and total number of genomic copies (in parentheses).
FIG 3
FIG 3
Retroviral envelope protein-based phylogenetic tree with the identified Ictidomys tridecemlineatus Env protein candidates. The maximum-likelihood tree inferred with the RaxML software was constructed using envelope amino acid sequences from mammalian endogenous retroviruses and from a series of infectious retroviruses. The horizontal branch length is proportional to the percentage of amino acid substitutions from the node (scale bar on the left), and the percent bootstrap values obtained from 1,000 replicates are indicated at the nodes. ALV, avian leukosis virus; BaEV, baboon endogenous virus; BERV, bovine endogenous retrovirus; FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; GaLV, gibbon ape leukemia virus; HERV, human endogenous retrovirus; HIV, human immunodeficiency virus; HTLV, human T cell leukemia virus; JSRV, Jaagsiekte sheep retrovirus; MMTV, murine mammary tumor virus; IAPE, intracisternal A-type particle with an envelope gene; MoMLV, Moloney murine leukemia virus; MPMV, Mason-Pfizer monkey virus; PERV, porcine endogenous retrovirus; RD114, feline endogenous type-C retrovirus.
FIG 4
FIG 4
Real-time qRT-PCR analysis of the candidate env gene transcripts from Marmota monax. Transcript levels in the indicated tissues are expressed as the ratio of the expression level of each env gene to that of the RPL19 control gene (see Materials and Methods). Tissue expression of the placenta-specific env1 gene is displayed in the top left panel; expression in the same tissues (abbreviated, displayed in the same order) is shown for the seven other env gene candidates.
FIG 5
FIG 5
Characterization of the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and woodchuck (Marmota monax) env-Mar1-containing endogenous retrovirus and of its integration site. (Upper panel) Structure of the env-Mar1-containing ERV and orthology between the ground squirrel and woodchuck env-Mar1 sequences. Repeated mobile elements (gray), proviral LTRs, and the degenerate gag-pol genes, as identified by the RepeatMasker and BLAST web programs, are positioned (the symbols used are given below the panel). Undetermined sequence is indicated by NNN. PCR primers used to identify the env-Mar1 orthologous copy in the woodchuck are indicated (black half arrows), and the regions of the determined sequences homologous to those of the ground squirrel are aligned. The spliced env subgenomic transcript as determined by 5′- and 3′-RACE of woodchuck placental RNA is indicated. (Lower panel) Absence of the env1-containing ERV in the genomes of distant mammalian lineages. The genomic locus of the Ictidomys tridecemlineatus env-Mar1-containing provirus (env in red), along with its surrounding Ceruloplasmin and TM4SF18 genes, was recovered from the UCSC Genome Browser (http://genome.ucsc.edu/), together with the syntenic loci of the indicated species genomes; the positions of exons (vertical lines) of the resident Ceruloplasmin and TM4SF18 genes and the sense of transcription (arrows) are indicated. Homology of the syntenic loci was analyzed using the MultiPipMaker alignment building tool. Homologous regions are shown as pale green boxes, and highly conserved regions (more than 100 bp without a gap displaying at least 70% identity) are shown as dark green boxes.
FIG 6
FIG 6
Primary sequences and alignment of the homologous ground squirrel and woodchuck Env-Mar1 protein. Primary amino acid sequence and characteristic structural features of the homologous Env-Mar1 proteins from the ground squirrel Ictidomys tridecemlineatus and from the woodchuck Marmota monax. The same color code and abbreviations as in Fig. 2 are used. Asterisks indicate amino acid identity, and colons indicate amino acid similarity.
FIG 7
FIG 7
Syncytin-Mar1 is a fusogenic retroviral envelope protein. (A) Assay for cell-cell fusion mediated by Syncytin-Mar1. The indicated cell lines were transfected with an expression vector for Syncytin-Mar1 or an empty vector (none), together with a LacZ expression vector. Cells were cultured for 1 to 2 days after transfection, fixed, and stained with X-Gal. Syncytia were detected in the syncytin-Mar1-transfected cells, with only mononucleated cells visible using the empty vector. Scale bar, 100 μm. (B) Assay for cell infection mediated by Syncytin-Mar1-pseudotyped virus particles. Pseudotypes were produced by cotransfection of human 293T cells with expression vectors for the MLV core, the Syncytin-Mar1 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. Virus titers assayed on a panel of target cells from human (293T, TE671, and SHSY-5Y), cat (G355.5), or rodent (208F and A23) expressed as focus-forming units (FFU) per ml ± the standard errors of the mean are corrected for the background values of control particles without an Env protein and are means from three independent experiments.
FIG 8
FIG 8
Structure of the placenta and in situ hybridization for syncytin-Mar1 expression on woodchuck placental sections. (A) Schematic representation (based on panel B) of the woodchuck placenta with, from fetus to mother, the labyrinthine zona (lz), the junctional zone (jz), and the maternal decidua (md). An enlarged view of a fetal villus tissue section stained with hematoxylin-eosin-saffron (HES) (upper right) shows the multinucleated syncytiotrophoblast (st) generated by fusion from the underlying mononucleated cytotrophoblast cells forming the interface between the fetal vessel (fv) and the maternal lacuna (ml). (B to J) Sections of placenta from woodchuck at midgestation, with the positions of the E to J panels indicated in panel A. (B, E, G, and I) HES staining of placental sections with the three layers (lz, jz, and md) indicated; in panel B the fetal villi (fvi) are delineated with a black dotted line, and the degenerating zone (dz) with a pink dotted line. (C, D, F, H, and J) In situ hybridization on serial sections observed at different magnifications, using digoxigenin-labeled antisense (C, F, H, and J) or sense (negative control [D]) riboprobes revealed with an alkaline phosphatase-conjugated anti-digoxigenin antibody. (E and F) Intermediate magnification of the labyrinth zone in the fetal villi delineated in panel A, with no detectable labeling. (G and H) Intermediate magnification of the labyrinth zone in formation delineated in panel A, with labeling close to the maternal lacunae. (I and J) Intermediate magnification of the junctional zone delineated in panel A, with labeling abutting the degenerating zone on the right. Scale bars: 0.5 mm for the serial sections (B to D), 50 μm for panels E and F, and 100 μm for panels G to J.
FIG 9
FIG 9
Status of syncytin-Mar1 during the radiation of the Sciuridae. Phylogenetic tree of the squirrel-related clade, with the families Sciuridae, Aplodontidae, and Gliridae, as well as the outgroup species Cavia porcellus and Mus musculus (dates and branching positions are from references , , , and 38), with the time scale at bottom left. Branches of the family Sciuridae whose interrelationships are still unresolved are represented as polytomies. The names of the species tested for the presence of the syncytin-Mar1 gene are indicated, together with the length (in amino acids) of the Syncytin-Mar1 proteins that were identified for each species (all of the sequences were deposited in GenBank under accession numbers KJ145238 to KJ145244, KJ670373, and KJ670374). Brackets indicate that only partial sequences could be retrieved. aa, amino acids; [c], coding sequence; [nc], noncoding sequence; –, no syncytin-Mar1 homologous sequence identified, by either PCR amplification or database search; nd, not done. The fusogenic activity of each cloned gene, as determined by the fusion assay in Fig. 7, is indicated.
FIG 10
FIG 10
Sequence conservation and evidence for purifying selection of syncytin-Mar1 in the tribe Marmotini. (Left) Maximum-likelihood phylogenetic tree determined using amino acid alignment of the Syncytin-Mar1 proteins identified in Fig. 9 and inferred with the RaxML software. The horizontal branch length and scale indicate the percentage of amino acid substitutions. The percent bootstrap values obtained from 1,000 replicates are indicated at the nodes. (Right) Double-entry table for the pairwise percentage of amino acid sequence identity between the Syncytin-Mar1 proteins among the indicated species (lower triangle) and the pairwise Nei-Gojobori dN/dS ratio (upper triangle). A color code is provided below the table for both series of values.
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References

    1. Black S, Arnaud F, Palmarini M, Spencer T. 2010. Endogenous retroviruses in trophoblast differentiation and placental development. Am. J. Reprod. Immunol. 64:255–264. 10.1111/j.1600-0897.2010.00860.x - DOI - PMC - PubMed
    1. Dupressoir A, Lavialle C, Heidmann T. 2012. From ancestral infectious retroviruses to bona fide cellular genes: role of the captured syncytins in placentation. Placenta 33:663–671. 10.1016/j.placenta.2012.05.005 - DOI - PubMed
    1. Lavialle C, Cornelis G, Dupressoir A, Esnault C, Heidmann O, Vernochet C, Heidmann T. 2013. Paleovirology of ‘syncytins,' retroviral env genes exapted for a role in placentation. Philos. Trans. R. Soc. Lond. B Biol. Sci. 368:20120507. 10.1098/rstb.2012.0507 - DOI - PMC - PubMed
    1. Blond JL, Lavillette D, Cheynet V, Bouton O, Oriol G, Chapel-Fernandes S, Mandrand B, Mallet F, Cosset FL. 2000. 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. J. Virol. 74:3321–3329. 10.1128/JVI.74.7.3321-3329.2000 - DOI - PMC - PubMed
    1. Mi S, Lee X, Li X, Veldman G, Finnerty H, Racie L, LaVallie E, Tang X, Edouard P, Howes S, Keith JJ, McCoy J. 2000. Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 17:785–789 - PubMed

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