An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard

Abstract

Syncytins are envelope genes from endogenous retroviruses that have been captured during evolution for a function in placentation. They have been found in all placental mammals in which they have been searched, including marsupials. Placental structures are not restricted to mammals but also emerged in some other vertebrates, most frequently in lizards, such as the viviparous Mabuya Scincidae. Here, we performed high-throughput RNA sequencing of a Mabuya placenta transcriptome and screened for the presence of retroviral env genes with a full-length ORF. We identified one such gene, which we named "syncytin-Mab1," that has all the characteristics expected for a syncytin gene. It encodes a membrane-bound envelope protein with fusogenic activity ex vivo, is expressed at the placental level as revealed by in situ hybridization and immunohistochemistry, and is conserved in all Mabuya species tested, spanning over 25 My of evolution. Its cognate receptor, required for its fusogenic activity, was searched for by a screening assay using the GeneBridge4 human/Chinese hamster radiation hybrid panel and found to be the MPZL1 gene, previously identified in mammals as a signal-transducing transmembrane protein involved in cell migration. Together, these results show that syncytin capture is not restricted to placental mammals, but can also take place in the rare nonmammalian vertebrates in which a viviparous placentotrophic mode of reproduction emerged. It suggests that similar molecular tools have been used for the convergent evolution of placentation in independently evolved and highly distant vertebrates.

Keywords: endogenous retrovirus; envelope protein; placenta; receptor; syncytin.

Fig. 1.

Phylogeny of vertebrates positioning mammals, the Mabuya lizard, and known syncytins. Mammals comprise the monotremes (e.g., platypus) still laying eggs, and the marsupials and eutherian mammals, which all possess a placenta (red font). The lizard Mabuya is shown in red font because it also possesses a placenta. A red arrow indicates the probable time of emergence of the mammalian placenta, which has been proposed to correspond to the primitive capture of an ancestral syncytin, thereafter replaced in evolution by the indicated present-day syncytins (reviewed in ref. 1). All currently described syncytin capture events are indicated by arrowheads in purple, together with the syncytin’s name. Branch length is proportional to time [expressed in My (15, 64, 65)], as indicated in the scale below the tree.

Fig. 2.

Structure of a canonical retroviral Env protein and characterization of the identified Mabuya 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 immunosuppressive domain (ISD) (blue), and the conserved C-X_5–7-C motif (CC) are indicated. (B) Characterization of the candidate Mabuya Env proteins. The hydrophobicity profile for each candidate is shown with the canonical structural features highlighted in A. (C) Retroviral envelope protein-based phylogenetic tree with the identified Mab-Env protein candidates in red. The unrooted PhyML maximum likelihood tree was obtained using TM subunit amino acid sequences (without the cytoplasmic tail) from syncytins (in blue) and a series of endogenous and infectious retroviruses (from the dataset in ref. 66). The horizontal scale bar below the tree represents the average number of substitutions per site, and the percent bootstrap values obtained from 1,000 replicates are indicated by circles on each branch (see key in figure). Retroviral families and major envelope types, the associated C-X_n-(C)C motif, and the presence or absence of an ISD are indicated on the tree. (D) Amino acid sequences and characteristic structural features of Mab-Env1/Mab-2 and Mab-Env3/Mab-4 (GenBank accession nos. MG254888–MG254891). Asterisks indicate amino acid identity; colons indicate amino acid similarity.

Fig. 3.

Real-time RT-qPCR analysis of candidate env gene expression in Mabuya sp. IV. Transcript levels are shown as the ratio between the expression levels of each env gene and those of the RPL19 control gene (SI Methods). Placental tissues and maternal ovary and oviduct tissues were recovered at two gestational stages (defined using ref. 33): stage 30, when the uterine syncytium starts to form in the placentome region, and stage 37, when the syncytium is well developed. The results for the four env candidate genes were obtained using the same series of tissues. Values are the means of duplicates from three samples ± SEM.

Fig. 4.

Characterization of the Mab-Env1–associated RT-containing pol gene and classification among known retroviral families. (A) ORF map of the identified genomic proviral fragment. Short bars indicate a start codon; tall bars indicate a stop codon. Green horizontal lines represent overlapping RNA-seq transcripts. Similarities to canonical retroviral genes by BLASTp search are colored in yellow for gag and purple for pol. Mab-Env1 is colored red. The conserved RT and TM domains used for the phylogenetic analyses in Figs. 2C and 4B are delineated. (B) Unrooted PhyML tree showing the position of retroviral endogenous and exogenous RT domains using the same dataset as in Fig. 2C when available (some syncytin-associated RTs were too degenerate to be identified). The Mab-Env1–associated RT is highlighted in red, and those associated with previously described syncytins are shown in blue. Major retroviral families are indicated on the tree. Bootstraps obtained after 1,000 replicates are indicated by circles on each branch (see key in figure). Branch length is proportional to the average number of substitutions per site (see scale). RT sequences are provided as Dataset S1.

Fig. 5.

Mab-Env1 is conserved only within the Mabuya genus. (Left) Species tree of Scincidae with A. carolinensis as an outgroup. Nodes with black circles are dated in Mya and placed proportionally to their age (28, 36, 64). Entry of Mab-Env1 is indicated by the white-tipped arrow; clades in which the envelope has been conserved are indicated by the black arrow. An asterisk indicates that a published genome is available (A. carolinensis). (Right) A plus sign indicates amplification of a Mab-Env1 internal 400-bp fragment (int) or the complete env ORF by genomic DNA PCR. Amplification of the complete ORF is restricted to the Mabuya genus, with no amplification in other Scincidae.

Fig. 6.

Structure of the Mabuya sp. IV placenta and expression pattern of Mab-Env1. (A) Schematic representation of the late-stage Mabuya sp. IV placenta (see ref. 27). (Left) Overview of a gravid uterus displaying the apposed maternal and fetal tissues. Maternal and fetal tissues are highly interdigitated in the placentome region. In addition to the placentome, the Mabuya placenta develops specialized regions for materno–fetal exchanges such as areolae and absorptive plaques. The brown/orange and gray/yellow areas represent the fetal and maternal tissues, respectively. (Right) Detailed scheme of the materno–fetal interface in the placentome and paraplacentome region. In the placentome, maternal and fetal tissues are highly interdigitated, with numerous microvilli between fetal and uterine cells. The fetal epithelium is formed by giant binucleated chorionic cells. The uterine epithelium is replaced by a large syncytial structure formed by the fusion of the uterine cells. In the paraplacentome the syncytium is abruptly replaced by uterine epithelium. Cells still present microvilli, but the tissues are no longer interdigitated. (B, Left) Hematoxylin eosin saffron (HES)-stained sections of placenta with the maternal uterine syncytium (ms), the maternal epithelium (me), and the fetal chorionic epithelium (fc) delineated; white arrowheads indicate maternal and black arrowheads indicate fetal blood vessels. (B, Right) In situ hybridization (odd rows) or immunohistochemistry (even rows) on serial sections for Mab-Env1 expression using digoxigenin-labeled antisense and sense riboprobes or an anti–Mab-Env1 mouse serum and preimmune serum. (Scale bars: 50 µm.) Areas marked by rectangles are enlarged on the right, and the maternal and fetal domains are delineated.

Fig. 7.

Mab-Env1 is a fusogenic retroviral envelope protein. (A) Schematic representation of the cell infection assay with Mab-Env–pseudotyped virus particles. Pseudotypes are produced by cotransfecting 293T cells with expression vectors for the MLV core, a β-galactosidase encoded by a nlsLacZ-containing retroviral transcript, and either a vector expressing Mab-Env proteins or a control vector. The supernatant of the transfected cells is then added to the indicated target cells, which are X-gal stained 3 d postinfection to reveal viral infection. (B) Panel of X-gal–stained target cells infected with particles without Env or pseudotyped with Mab-Env1, Mab-Env2, or the Env protein from A-MLV as positive control. (C) Quantification of viral titers expressed in focus-forming units per milliliter after infection with Mab-Env1–pseudotyped MLV virions. Values shown are the mean of three independent experiments ± SD. (D) Schematic representation of the cell–cell fusion assay. 293T cells were cotransfected with a plasmid expressing a nuclear β-galactosidase and a plasmid expressing either Mab-Env1 or a control plasmid. After 48 h a 5-min acidic shock was performed (or not) using Dulbecco’s phosphate-buffered saline (DPBS)⋅HCl at either pH 4 or 7. Cells were X-gal stained 6 h afterward. (E) Mab-Env1 is able to induce cell–cell fusion after an acidic shock. After exposition to acid medium, cells transfected with the Mab-Env1–expressing plasmid show a degree of fusion equivalent to that of cells transfected with a plasmid expressing the Env protein of mouse mammary tumor virus (MMTV), which was used as a positive control.

Fig. 8.

Structure and expression of the Mab-Env1 receptor MPZL1. (A) Alignment of Mabuya MPZL1 (GenBank accession no. MG254887) and human MPZL1 amino acid sequences with major domains and sites indicated. Asterisks indicate amino acid identities, and colons indicate amino acid similarities. (B) To-scale schematic representation of the mature human MPZL1 with major domains indicated. (C) Human and Mabuya MPZL1 act as receptors for Mab-Env1–mediated infection. Target A23 cells were transfected with a plasmid expressing human or Mabuya MPZL1 or an empty vector. Twenty-four hours posttransfection, cells were infected using MLV particles carrying an nlsLacZ reporter gene and pseudotyped with Mab-Env1 or Mab-Env2 or without Env. Cells were X-gal stained 72 h after infection, and infection foci were quantified. Values shown are the mean of three independent experiments ± SD. (D) MPZL1 is expressed in a wide range of tissues. RT-qPCR analysis of MPZL1 expression levels was performed in the same way and on the same series of tissues as in Fig. 3. (E) MPZL1 is expressed at the materno–fetal interface. (Left) HES-stained sections of placenta with the maternal syncytium (ms), the maternal epithelium (me), and the fetal chorionic epithelium (fc) delineated; white arrowheads indicate maternal and black arrowheads indicate fetal blood vessels. (Right) In situ hybridization on serial sections for MPZL1 expression using digoxigenin-labeled antisense and sense riboprobes. (Scale bars: 50 µm.) Areas marked by rectangles are enlarged on the right, and the maternal and fetal domains are delineated.

Fig. 9.

Mab-Env1 induces degradation and phosphorylation of MPZL1. MCF10A cells were transduced either with an empty lentiviral vector or with the same vector coding for syncytin-Car1 or Mab-Env1. Con A treatment was performed (or not) 1 h before cell lysis as a positive control. Lysates were deglycosylated using PNGase F, and Western blot analysis was performed using an antibody recognizing both phosphorylated and unphosphorylated MPZL1 (Top) or only phosphorylated MPZL1 (Middle). (Bottom) An anti–tubulin-γ antibody was used to compare total protein levels in each well. Each set of blots represents an independent set of transduced cells.