Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials

Abstract

Syncytins are genes of retroviral origin captured by eutherian mammals, with a role in placentation. Here we show that some marsupials-which are the closest living relatives to eutherian mammals, although they diverged from the latter ∼190 Mya-also possess a syncytin gene. The gene identified in the South American marsupial opossum and dubbed syncytin-Opo1 has all of the characteristic features of a bona fide syncytin gene: It is fusogenic in an ex vivo cell-cell fusion assay; it is specifically expressed in the short-lived placenta at the level of the syncytial feto-maternal interface; and it is conserved in a functional state in a series of Monodelphis species. We further identify a nonfusogenic retroviral envelope gene that has been conserved for >80 My of evolution among all marsupials (including the opossum and the Australian tammar wallaby), with evidence for purifying selection and conservation of a canonical immunosuppressive domain, but with only limited expression in the placenta. This unusual captured gene, together with a third class of envelope genes from recently endogenized retroviruses-displaying strong expression in the uterine glands where retroviral particles can be detected-plausibly correspond to the different evolutionary statuses of a captured retroviral envelope gene, with only syncytin-Opo1 being the present-day bona fide syncytin active in the opossum and related species. This study would accordingly recapitulate the natural history of syncytin exaptation and evolution in a single species, and definitely extends the presence of such genes to all major placental mammalian clades.

Keywords: endogenous retrovirus; envelope protein; fusogenic activity; marsupials; syncytiotrophoblast.

Fig. 1.

Phylogeny of mammals and previously identified syncytin genes. Mammals comprise the monotremes, the marsupials, and the eutherians, the latter comprising the four major clades: Afrotheria (I), Xenarthra (II), Euarchontoglires (III), and Laurasiatheria (IV) (data from ref. 1). Branch length is proportional to time (in My), and the time of insertion of the different syncytins identified to date is indicated with arrowheads.

Fig. 2.

Structure of a canonical retroviral Env protein and characterization of the identified opossum 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), and the putative ISD (blue) along with the conserved C-X_5/6/7-C motif are indicated. (B) Characterization of the candidate opossum (M. domestica) Env proteins. (B, Left) The hydrophobicity profile of each candidate is shown with the canonical structural features highlighted in A positioned, when present (same color code). (B, Right) Number of full-length env gene ORFs within each family of element and total number of genomic copies (in parentheses). (C) Retroviral Env protein-based phylogenetic tree with the identified Opo-Env protein candidates. Env proteins that were grouped into single families of elements are distinguished by indication of their chromosomal position (Table S1). A maximum-likelihood tree using TM subunit amino acid sequences from syncytins and a series of endogenous and infectious retroviruses is shown. 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 >50% (obtained from 1,000 replicates) are indicated at the nodes. ALV, avian leukemia virus; MPMV, Mason–Pfizer monkey virus; Env-Cav1, “syncytin-like” Cavia porcellus Env1 protein; KoRV, koala retrovirus; GaLV, gibbon ape leukemia virus; PERV, porcine endogenous retrovirus; BaEV, baboon endogenous virus; HIV1, HIV type 1; HERV, human endogenous retrovirus; MoMLV, Moloney murine leukemia virus; HTLV-2, human T-lymphotropic virus type 2; BLV, bovine leukemia virus; JSRV, Jaagsiekte retrovirus; MMTV, murine mammary tumor virus; mIAPE, M. musculus intracisternal A-type particle with an Env gene; FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; RD114, feline endogenous type-C retrovirus.

Fig. 3.

Real-time qRT-PCR analysis of the candidate opo-env gene transcripts from the opossum (M. domestica). Transcript levels are expressed as the ratio of the expression level of each opo-env gene to that of the RPLP0 control gene (SI Methods). Because of the high interpenetration of maternal and fetal tissues, placental and uterine tissues are analyzed as a whole at three gestational dates (days 12, 13, and 14). The results obtained with the same series of tissues for the nine env gene candidates are shown. Values are the means of duplicates from three samples ± SEM.

Fig. 4.

Structure of the opossum (M. domestica) placenta and ISH for opo-env1 and -env3 expression on placental sections. (A) Schematic representation of the opossum placenta. (A, Left) Overview of a gravid uterus displaying the apposed maternal and fetal tissues. The yellow and gray areas represent the fetus and mother tissues, respectively. (A, Right) Detailed scheme of the definitive placental structure. The trilaminar placenta (fetal endoderm, fetal vessels, and syncytiotropholast layer) is apposed to the maternal uterine epithelium. Locally, the maternal epithelium is invaded by projections from the syncytiotrophoblast layer that directly contact the maternal vessels. A large trabecular meshwork of uterine glands can be seen underneath the maternal uterine epithelium. (B) Semithin section of the feto-maternal interface, corresponding to the area boxed in A, with the various constituents delineated on the right. The multinucleate syncytiotrophoblast (st, yellow) penetrates the uterine epithelium (ue, dark gray) and contacts the maternal vessel (mv, pink). (C) hematoxylin eosin saffron (HES)-stained sections of placenta and ISH on serial sections for opo-env1 (Upper) or opo-env3 (Lower) placental transcripts using digoxigenin-labeled antisense or sense riboprobes revealed with an alkaline phosphatase-conjugated antidigoxigenin antibody. (C, Upper) Placental villi. (C, Lower) Uterine glands. Specific staining is observed at the level of the feto-maternal interface of placental villi for opo-env1 and at the level of the maternal uterine glands for opo-env3 (enlarged views in Right). (Scale bar: 10 µm.)

Fig. 5.

Syncytin-Opo1 is a fusogenic retroviral Env protein. (A) Schematic representation of the coculture assay for cell–cell fusion with Syncytin-Opo1. Human 293T cells were transfected with an expression vector for Syncytin-Opo1 (or an empty “No Env” vector as a control) and a plasmid expressing a nuclear beta-galactosidase (nlsLacZ). After transfection, the 293T cells were cocultured with target cells and X-gal–stained 48 h later. (B) Syncytium formation (see arrows) with the indicated Syncytin-Opo1, using A23 cells as the target (no syncytium with the No Env control). (Scale bars: 200 µm.)

Fig. 6.

Characterization of the opossum (M. domestica) syncytin-Opo1 gene and its proviral integration site. (A) Structure of the syncytin-Opo1 proviral sequence and integration site. Repeated mobile elements as identified by the RepeatMasker web program are positioned. Of note, the provirus shows signs of degeneration, with a deletion within the 3′ LTR and degenerate gag-pol sequences, with the noticeable exception of the syncytin-opo1 gene. PCR primers used to identify the syncytin-Opo1 orthologous copy in other Monodelphis species are indicated (black half arrows). The spliced env subgenomic transcript as determined by RACE-PCR of opossum placental RNA is indicated (GenBank accession no. KM235357). Putative GCM1 binding sites are indicated as blue vertical lines. Sequence of the integration site of syncytin-Opo1, showing evidence for retroviral integration, is provided above the schematized provirus: characteristic sequences are shown, including LTRs (with TG . . . CA borders), PBS for a proline tRNA, and TSD (red boxes). (B) Absence of Syncytin-Opo1 in the genomes of representative species of mammalian lineages. The genomic locus of syncytin-Opo1 (with the env gene in red), along with the surrounding MICAL3 and BCL2L13 genes were recovered from the UCSC Genome Browser (genome.ucsc.edu/), together with the syntenic loci of marsupials (i.e., wallaby, and Tasmanian devil) and eutherian mammals genomes; exons (vertical lines) of the MICAL3 and BCL2L13 genes and the sense of transcription (arrows) are indicated. Homology of the syntenic loci was analyzed by using the MultiPipMaker alignment building tool. Homologous regions are shown as pale green boxes, and highly conserved regions (>100 bp without a gap displaying at least 70% identity) are shown as dark green boxes.

Fig. 7.

Insertion date and conservation of syncytin-Opo1, pan-Mars-env2, and opo-env3 during marsupial evolution. (Left) Marsupial phylogenetic tree (data from refs. and –40). Horizontal branch length is proportional to time (scale bar on top), with the exception of the Monodelphis lineage where no information is available to date. The names of the 26 marsupial species tested are indicated, together with the names of the Ameridelphia and Australidelphia corresponding orders (Das., Dasyuromorphia; Per., Peramelemorphia). Asterisks indicate species whose genome is available in genomic databases. (Right) The length (in amino acids) of the different env genes that could be identified is indicated. [nc], presence of a full-length noncoding syncytin-Opo1 gene; +/−, multiple homologous mixed sequences; −, no env homologous sequence identified, by either PCR-amplification or database search. The fusion activity for each syncytin-Opo1 cloned gene, as determined by the cell–cell fusion assay, is indicated. nr, not relevant. Arrows indicate the respective insertion dates of the different env genes.

Fig. 8.

Characterization of the marsupial pan-Mars-env2 gene and of its proviral integration site. (A) Structure of the opossum (M. domestica), Tasmanian devil (S. harrisii), and wallaby (M. eugenii) env2 proviral sequences and integration sites. Repeated mobile elements as identified by the RepeatMasker Web program are positioned. Of note, the proviruses show signs of degeneration, with no LTR identifiable and highly degenerate gag-pol sequences. PCR primers used to identify the env2 orthologous copies in marsupial species are indicated (black half arrows). (B) Demonstration of the orthology of env2 (dubbed pan-Mars-env2) in the genomes of sequenced marsupial species. The genomic locus of the opossum pan-Mars-env2 (in red), along with the surrounding FUT10 and PRSS12 genes, was recovered from the UCSC Genome Browser (genome.ucsc.edu/), together with the syntenic loci of the wallaby and the Tasmanian devil genomes. The genome of wallaby is incompletely assembled, and the FUT10 and PRSS12 genes are located on two distinct unassembled scaffolds, with pan-Mars-env2 being located on the scaffold containing the PRSS12 gene. The FUT10 and PRSS12 genes were not located on the same chromosome in other eutherian mammalian species, preventing syntenic comparisons. Same color code and symbols are used as in Fig. 6. (C) Sequence conservation and evidence for purifying selection pan-Mars-Env2 in marsupials. (C, Left) pan-Mars-Env2 phylogenetic tree determined using amino acid alignment of the encoded proteins identified in Fig. 7, by the maximum-likelihood method. 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. (C, Right) Double-entry table for the pairwise percentage of amino acid sequence identity between the pan-Mars-env2 gene among the indicated species (lower triangle), and the pairwise Nei–Gojobori nonsynonymous to synonymous mutation rate ratio (dN/dS; upper triangle). A color code is provided for both series of values.