Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV <i>Syncytin</i>, Captured for a Function in Placentation

<div><p><i>Syncytins</i> are envelope genes of retroviral origin that have been co-opted for a role in placentation. They promote cell–cell fusion and are involved in the formation of a syncytium layer—the syncytiotrophoblast—at the materno-fetal interface. They were captured independently in eutherian mammals, and knockout mice demonstrated that they are absolutely required for placenta formation and embryo survival. Here we provide evidence that these “necessary” genes acquired “by chance” have a definite lifetime with diverse fates depending on the animal lineage, being both gained and lost in the course of evolution. Analysis of a retroviral envelope gene, the <i>envV</i> gene, present in primate genomes and belonging to the endogenous retrovirus type V (ERV-V) provirus, shows that this captured gene, which entered the primate lineage >45 million years ago, behaves as a <i>syncytin</i> in Old World monkeys, but lost its canonical fusogenic activity in other primate lineages, including humans. In the Old World monkeys, we show—by <i>in situ</i> analyses and <i>ex vivo</i> assays—that <i>envV</i> is both specifically expressed at the level of the placental syncytiotrophoblast and fusogenic, and that it further displays signs of purifying selection based on analysis of non-synonymous to synonymous substitution rates. We further show that purifying selection still operates in the primate lineages where the gene is no longer fusogenic, indicating that degeneracy of this ancestral <i>syncytin</i> is a slow, lineage-dependent, and multi-step process, in which the fusogenic activity would be the first canonical property of this retroviral envelope gene to be lost.</p> </div

EnvV2 protein expression and cell surface localization.

<p>(A) Cell-cell fusion assay for hemagglutinin (HA)-tagged EnvV2 proteins in G355.5 cells. Transient transfection of feline cells was assayed with a control plasmid (none) or plasmids expressing the EnvV2 protein, tagged (or not) with the HA epitope. May-Grünwald-Giemsa staining was carried out 24 h posttransfection and fusion index measured as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003400#pgen-1003400-g003" target="_blank">Figure 3</a>. (B) Cell-surface expression of EnvV2 proteins. A23 cells were transfected with the indicated EnvV2-HA expression vectors. The cell surface-expressed proteins were biotinylated for further purification (see Methods). Detection of the EnvV2 proteins was performed by Western blotting of the two cellular fractions obtained –<i>i.e.</i> the intracellular cell lysates (I) and the cell surface biotinylated proteins (S)- with a monoclonal anti-HA antibody (upper). As a control for the fractioning, the blot was hybridized with a monoclonal antibody against the GAPDH soluble cellular protein (lower).</p

qRT–PCR analysis of <i>envV2</i> transcripts in macaque and human tissues.

<p>Real-time qRT-PCR analysis of the macaque and human <i>envV2</i> transcripts in a panel of 17 macaque (upper) and human (lower) tissues, with transcript expressed as percent of maximum, after normalization with a control gene (PPIA) mRNA (see Methods).</p

Cell–cell fusion assay for the primate EnvV proteins.

<p>(A) Env-expressing vectors and rationale of the assay. Each of the 24 cloned <i>envV</i> genes was introduced into the phCMV expression vector, between the beta-globin intron and polyadenylation (pA) sequences. Cells were transfected and stained with May-Grünwald-Giemsa 24 h after transfection. The fusion index is defined as [(<i>N</i>-<i>S</i>)/<i>T</i>]×100, where <i>N</i> is the number of nuclei in the syncytia, <i>S</i> is the number of syncytia, and <i>T</i> is the total number of nuclei counted. (B) 293T cells transfected with expression vectors for no protein (none), and for the human, macaque and marmoset EnvV2 (V2 hum, V2 mac and V2 mar, respectively), displaying large multinucleated syncytia 24 h later, only upon transfection with the latter two. (C) Histogram showing the fusion index of the indicated series of primate <i>envV1</i> (upper) and <i>envV2</i> (lower) genes in 293T cells transfected with the corresponding expression vectors. The primate phylogeny is illustrated below, with the Old World monkeys (OWM) and New World monkeys (NWM) indicated. None, no protein; hum, human; cpz, chimpanzee; gor, gorilla; gib, gibbon; mac, macaque; bab, baboon; agm, African green monkey; lan, langur; mar, marmoset; tam, tamarin; sak, saki. (D) Same as (C) but with feline G355.5 cells transfected with the <i>envV2</i> expression vectors.</p

Structure of a canonical retroviral envelope protein and characterization of the <i>envV1</i> and <i>envV2</i> genes in primates.

<p>(A) Schematic representation of a retroviral Env protein, delineating the surface (SU) and transmembrane (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 immunosuppressive domain (ISD) (blue) along with the conserved C-C motif are indicated. (B) Genomic organization of the HERV-V1 and HERV-V2 proviruses. The retroviral <i>env</i> ORF (red open box) and long terminal repeats (LTRs; arrowed open boxes), and the Alu (light gray boxes) and MER50 (dark gray boxes) retroelements are indicated. Positions of the primers designed to amplify the <i>env</i> coding sequences are indicated. (C) ORF map of the cloned <i>envV</i> genes. The dark gray boxes delineate the envelope coding sequences and the light gray boxes represent the ORFs still present downstream of stop codons or frameshifts. The deletion in the orangutan <i>envV1</i> gene is depicted by an open triangle.</p

<i>In situ</i> hybridization for <i>envV2</i> expression in the macaque and human placenta.

<p>(A) Schematic representation of the simian placenta, with an enlarged villus bathed in maternal blood and displaying –from the maternal to the fetal side- the syncytiotrophoblast (ST) layer, the mononucleated cytotrophoblasts (CT) and the fetal vessels. (B) Serial sections of macaque (upper row) and human (two lower rows) placenta. (columns 1–3) <i>in situ</i> hybridization with digoxigenin-labeled <i>envV2</i> sense (negative control, column 1) and antisense (columns 2,3) riboprobes, revealed with an alkaline phosphatase-conjugated anti-digoxigenin antibody. (column 4) immunohistochemical staining of the Ki67 nuclear antigen. (columns 3,4) enlarged views with the empty arrowheads pointing to the syncytiotrophoblast positively stained for <i>envV2</i> mRNA, and the filled arrows to Ki67-marked mononucleated cytotrophoblasts.</p

APOBEC3 family members and their associated roles in exogenous viruses and endogenous retroelements restriction

Data are compiled from [27, 77, 87, 90, 126-140].<p><b>Copyright information:</b></p><p>Taken from "Uracil within DNA: an actor of antiviral immunity"</p><p>http://www.retrovirology.com/content/5/1/45</p><p>Retrovirology 2008;5():45-45.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2427051.</p><p></p

Branch-specific analysis of selection along the <i>envV2</i>-based phylogenetic tree.

<p>Analysis was performed using the GA-branch method from the HyPhy Package on the webserver <a href="http://www.datamonkey.org" target="_blank">www.datamonkey.org</a>, and the selected model was the one with the best Akaike Information Criterion (AIC). Branch-specific analysis allows the non-synonymous to synonymous mutation ratio (dN/dS) to vary between phylogenetic branches. Such an analysis on the <i>envV2</i> gene reveals that all the branches are under strong purifying selection (dN/dS<1) with no significant difference in the strength of selection between New World monkeys (NWM), Old World monkeys (OWM) and hominoids. Left: maximum likelihood tree of <i>envV2</i> (same as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003400#pgen-1003400-g008" target="_blank">Figure 8</a>) is represented together with the branch numbers. The color code for each branch class is indicated on top. Right: estimated branch-specific dN/dS values are indicated ± standard deviation.</p

Biosynthesis pathways of ribonucleotides and deoxyribonucleotides in mammalian cells and the possible consequence of the misincorporation and repair of uracil residues in DNA

synthesis of AMP, CMP, GMP and UMP ribonucleotides allows the formation of dATP, dCTP, dGTP, dTTP and dUTP deoxyribonucleotides, which can be readily incorporated in DNA by cellular DNA polymerases. Note that dTTP derives from dUTP hydrolysis. Abbreviations: A, adenine; C, cytosine; G, guanine; T, thymine; U, uracil; MP, monophosphate; DP, diphosphate; TP, triphosphate; rNDP, ribonucleotide diphosphate; NMPK, nucleotide monophosphate kinase; NDPK, nucleotide diphosphate kinase.<p><b>Copyright information:</b></p><p>Taken from "Uracil within DNA: an actor of antiviral immunity"</p><p>http://www.retrovirology.com/content/5/1/45</p><p>Retrovirology 2008;5():45-45.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2427051.</p><p></p

Phylogenetic tree of primates, and status of the captured endogenous retrovirus envelope genes with placental expression.

<p>The date of insertion of the indicated retroviral envelope genes into the genome of primate ancestors is indicated for the <i>syncytin</i> genes (<i>i.e.</i> the EnvFRD- and EnvW-encoding genes, in red <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003400#pgen.1003400-Blaise1" target="_blank">[8]</a> and blue <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003400#pgen.1003400-Bonnaud1" target="_blank">[6]</a>, respectively, and for the EnvV- and EnvR-encoding genes, in orange <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003400#pgen.1003400-Kjeldbjerg1" target="_blank">[27]</a> and green <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003400#pgen.1003400-Herv1" target="_blank">[26]</a>, respectively). Their presence in the various primate lineages is indicated with filled squares when the gene still possesses a full-length ORF, with hatched squares when the coding sequence is prematurely interrupted, and an empty square when the gene is no longer present. In addition, fusogenic activities (as determined by ex vivo cell-cell fusion assays in ref <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003400#pgen.1003400-Mallet1" target="_blank">[5]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003400#pgen.1003400-Blaise1" target="_blank">[8]</a>) are schematized with an upper triangle when present, and immunosuppressive activities (as assayed in ref <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003400#pgen.1003400-Mangeney1" target="_blank">[19]</a>) with lower triangles. Branch length is proportional to time (in million years).</p