Restriction by APOBEC3 proteins of endogenous retroviruses with an extracellular life cycle: ex vivo effects and in vivo "traces" on the murine IAPE and human HERV-K elements

<p>Abstract</p> <p>Background</p> <p>APOBEC3 cytosine deaminases have been demonstrated to restrict infectivity of a series of retroviruses, with different efficiencies depending on the retrovirus. In addition, APOBEC3 proteins can severely restrict the intracellular transposition of a series of retroelements with a strictly intracellular life cycle, including the murine IAP and MusD LTR-retrotransposons.</p> <p>Results</p> <p>Here we show that the IAPE element, which is the infectious progenitor of the strictly intracellular IAP elements, and the infectious human endogenous retrovirus HERV-K are restricted by both murine and human APOBEC3 proteins in an <it>ex vivo </it>assay for infectivity, with evidence in most cases of strand-specific G-to-A editing of the proviruses, with the expected signatures. <it>In silico </it>analysis of the naturally occurring genomic copies of the corresponding endogenous elements performed on the mouse and human genomes discloses "traces" of APOBEC3-editing, with the specific signature of the murine APOBEC3 and human APOBEC3G enzymes, respectively, and to a variable extent depending on the family member.</p> <p>Conclusion</p> <p>These results indicate that the IAPE and HERV-K elements, which can only replicate via an extracellular infection cycle, have been restricted at the time of their entry, amplification and integration into their target host genomes by definite APOBEC3 proteins, most probably acting in evolution to limit the mutagenic effect of these endogenized extracellular parasites.</p

Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new "syncytin" in a third order of mammals

Abstract Background Syncytins are envelope genes of retroviral origin that have been co-opted by the host to mediate a specialized function in placentation. Two of these genes have already been identified in primates, as well as two distinct, non orthologous genes in rodents. Results Here we identified within the rabbit Oryctolagus cuniculus-which belongs to the lagomorpha order- an envelope (env) gene of retroviral origin with the characteristic features of a bona fide syncytin, that we named syncytin-Ory1. An in silico search for full-length env genes with an uninterrupted open reading frame within the rabbit genome first identified two candidate genes that were tested for their specific expression in the placenta by quantitative RT-PCR of RNA isolated from a large set of tissues. This resulted in the identification of an env gene with placenta-specific expression and belonging to a family of endogenous retroelements present at a limited copy number in the rabbit genome. Functional characterization of the identified placenta-expressed env gene after cloning in a CMV-driven expression vector and transient transfection experiments, demonstrated both fusogenic activity in an ex vivo cell-cell fusion assay and infectivity of pseudotypes. The receptor for the rabbit syncytin-Ory1 was found to be the same as that for human syncytin-1, i.e. the previously identified ASCT2 transporter. This was demonstrated by a co-culture fusion assay between hamster A23 cells transduced with an expression vector for ASCT2 and A23 cells transduced with syncytin-Ory1. Finally, in situ hybridization of rabbit placenta sections with a syncytin-Ory1 probe revealed specific expression at the level of the junctional zone between the placental lobe and the maternal decidua, where the invading syncytial fetal tissue contacts the maternal decidua to form the labyrinth, consistent with a role in the formation of the syncytiotrophoblast. The syncytin-Ory1 gene is found in Leporidae but not in Ochotonidae, and should therefore have entered the lagomorpha order 12-30 million years ago. Conclusion The identification of a novel syncytin gene within a third order of mammals displaying syncytiotrophoblast formation during placentation strongly supports the notion that on several occasions retroviral infections have resulted in the independent capture of genes that have been positively selected for a convergent physiological role.</p

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

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

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