We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the “mechanical” function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be “switched off” by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.
Syncytins 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 envV 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 syncytin in Old World monkeys, but lost its canonical fusogenic activity in other primate lineages, including humans. In the Old World monkeys, we show—by in situ analyses and ex vivo assays—that envV 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 syncytin 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.
Syncytins are “new” genes encoding the envelope protein of captured endogenous retroviral elements. Their unambiguous status of “cellular gene” was recently demonstrated by knocking them out in genetically modified mice, showing their absolute requirement for placenta formation and embryo survival, via formation by cell–cell fusion of the feto-maternal syncytium interface. These genes are remarkable, as they are “necessary” for a basic function in placental mammals and yet they were acquired “by chance” on multiple occasions and independently in diverse mammalian species. We proposed that syncytins have been pivotal for the emergence of animals with a placenta from those laying eggs via the capture of a founding retroviral env gene, then subsequently replaced in the diverse mammalian lineages upon successive and independent germline infections by new retroviruses and co-optation of their env gene, each new gene providing its host with a positive selective advantage. This hypothesis would account for the diversity of the captured syncytins that can be currently found, concomitant with the diversity of placental architectures. A consequence of this paradigm is that evidence for “decaying syncytins” in eutherian mammals should exist, and this is precisely what we sought—and found—in this study.
The IAPE (Intracisternal A-type Particles elements with an Envelope) family of murine endogenous retroelements is present at more than 200 copies in the mouse genome. We had previously identified a single copy that proved to be fully functional, i.e. which can generate viral particles budding out of the cell and infectious on a series of cells, including human cells. We also showed that IAPE are the progenitors of the highly reiterated IAP elements. The latter are now strictly intracellular retrotransposons, due to the loss of the envelope gene and re-localisation of the associated particles in the course of evolution. In the present study we searched for the cellular receptor of the IAPE elements, by using a lentiviral human cDNA library and a pseudotype assay on transduced cells. We identified Ephrin A4, a GPI-anchored molecule involved in several developmental processes, as a receptor for the IAPE pseudotypes. We also found that the other 4 members of the Ephrin A family –but not those of the closely related Ephrin B family- were also able to mediate IAPE cell entry, thus significantly increasing the amount of possible cell types susceptible to IAPE infection. We show that these include mouse germline cells, as illustrated by immunohistochemistry experiments, consistent with IAPE genomic amplification by successive re-infection. We propose that the uncovered properties of the identified receptors played a role in the accumulation of IAPE elements in the mouse genome, and in the survival of a functional copy.
In mammals, nearly half the genome is composed of reiterated scattered sequences. Some of them, called endogenous retroviruses, have a structure similar to that observed for the integrated form of infectious retroviruses. The current theory to account for their presence is that an infectious retrovirus once infected the germline of its host. This viral genome was then transmitted to the progeny and expressed from there, producing new infectious particles, which could re-infect new germline cells and thus increase the viral genomic copy number. However no evidence has yet been provided to support this model. In this study, we identify a family of five cellular proteins, the Ephrin As, as receptors for a model mouse family of endogenous retroviruses, the IAPE elements. We analyse their expression pattern and show that both the oocytes and some male germline cells express Ephrin A proteins and can thus be infected by IAPE particles. This finding strongly supports the current model of ERVs amplification. In addition, the IAPE envelope ability to use five different cellular receptors suggests that it might be impossible for the host to evolve a resistance against this viral element, and provides a clue on how the IAPE family survived so long in the mouse genome.
The ability of mammalian cytidine deaminases encoded by the APOBEC3 (A3) genes to restrict a broad number of endogenous retroelements and exogenous retroviruses, including murine leukemia virus and human immunodeficiency virus (HIV)-1, is now well established. The RNA editing family member apolipoprotein B (apo B)-editing catalytic subunit 1 (APOBEC1; A1) from a variety of mammalian species, a protein involved in lipid transport and which mediates C–U deamination of mRNA for apo B, has also been shown to modify a range of exogenous retroviruses, but its activity against endogenous retroelements remains unclear. Here, we show in cell culture-based retrotransposition assays that A1 family proteins from multiple mammalian species can also reduce the mobility and infectivity potential of LINE-1 (long interspersed nucleotide sequence-1, L1) and long-terminal repeats (LTRs) retrotransposons (or endogenous retroviruses), such as murine intracisternal A-particle (IAP) and MusD sequences. The anti-L1 activity of A1 was mainly mediated by a deamination-independent mechanism, and was not affected by subcellular localization of the proteins. In contrast, the inhibition of LTR-retrotransposons appeared to require the deaminase activity of A1 proteins. Thus, the AID/APOBEC family proteins including A1s employ multiple mechanisms to regulate the mobility of autonomous retrotransposons in several mammalian species.
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.
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.
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.
Detection of nucleic acids and induction of type I interferons (IFNs) are principal elements of antiviral defense, but can cause autoimmunity if misregulated. Cytosolic DNA detection activates a potent, cell-intrinsic antiviral response through a poorly defined pathway. In a screen for proteins relevant to this interferon-stimulatory DNA (ISD) response, we identify 3’ repair exonuclease 1 (Trex1). Mutations in the human trex1 gene cause Aicardi-Goutieres Syndrome (AGS) and chilblain lupus, but the molecular basis of these diseases is unknown. We define Trex1 as an essential negative regulator of the ISD response and delineate the genetic pathway linking Trex1 deficiency to lethal autoimmunity. We show that single-stranded DNA derived from endogenous retroelements accumulates in Trex1-deficient cells and that Trex1 can metabolize reverse-transcribed DNA. These findings reveal a cell-intrinsic mechanism for initiation of autoimmunity, implicate the ISD pathway as the cause of AGS, and suggest an unanticipated contribution of endogenous retroelements to autoimmunity.
Several families of endogenous retroviruses (ERVs) have been identified in the mouse genome, in several instances by in silico searches, but for many of them it remains to be determined whether there are elements that can still encode functional retroviral particles. Here, we identify, within the GLN family of highly reiterated ERVs, one, and only one, copy that encodes retroviral particles prone to infection of mouse cells. We show that its envelope protein confers an ecotropic host range and recognizes a receptor different from mCAT1 and mSMIT1, the two previously identified receptors for other ecotropic mouse retroviruses. Electron microscopy disclosed viral particle assembly and budding at the cell membrane, as well as release of mature particles into the extracellular space. These particles are closely related to murine leukemia virus (MLV) particles, with which they have most probably been confused in the past. This study, therefore, identifies a new class of infectious mouse ERVs belonging to the family Gammaretroviridae, with one family member still functional today. This family is in addition to the two MLV and mouse mammary tumor virus families of active mouse ERVs with an extracellular life cycle.
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.
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 ex vivo assay for infectivity, with evidence in most cases of strand-specific G-to-A editing of the proviruses, with the expected signatures. In silico 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.
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.
Viruslike particles which displayed a peculiar wheellike appearance that distinguished them from A-, B- or C-type particles had previously been described in the early mouse embryo. The maximum expression of these so-called epsilon particles was observed in two-cell-stage embryos, followed by their rapid decline at later stages of development and no particles detected at the zygote one-cell stage. Here, we show that these particles are in fact produced by a newly discovered murine endogenous retrovirus (ERV) belonging to the widespread family of mammalian ERV-L elements and named MuERV-L. Using antibodies that we raised against the Gag protein of these elements, Western blot analysis and in toto immunofluorescence studies of the embryos at various stages disclosed the same developmental expression profile as that observed for epsilon particles. Using expression vectors for cloned, full-length, entirely coding MuERV-L copies and cell transfection, direct identification of the epsilon particles was finally achieved by high-resolution electron microscopy.
Human trophoblast expresses two fusogenic retroviral envelope proteins, the widely studied syncytin 1, encoded by HERV-W and the recently characterized syncytin 2 encoded by HERV-FRD. Here we studied syncytin 2 in normal and Trisomy 21-affected placenta associated with abnormal trophoblast differentiation. Syncytin 2 immunolocalization was restricted throughout normal pregnancy to some villous cytotrophoblastic cells (CT). During the second trimester of pregnancy, syncytin 2 was immunolocalized in some cuboidal CT in T21 placentas, whereas in normal placentas it was observed in flat CT, extending into their cytoplasmic processes. In vitro, CT isolated from normal placenta fuse and differentiate into syncytiotrophoblast. At the same time, syncytin 2 transcript levels decreased significantly with syncytiotrophoblast formation. In contrast, CT isolated from T21-affected placentas fused and differentiated poorly and no variation in syncytin 2 transcript levels was observed. Syncytin 2 expression illustrates the abnormal trophoblast differentiation observed in placenta of fetal T21-affected pregnancies.
We had previously identified active autonomous copies of the MusD long terminal repeat-retrotransposon family, which have retained transpositional activity. These elements are closely related to betaretroviruses but lack an envelope (env) gene. Here we show that these elements encode strictly intracellular virus-like particles that can unambiguously be identified by electron microscopy. We demonstrate intracellular maturation of the particles, with a significant proportion of densely packed cores for wild-type MusD but not for a protease mutant. We show that the molecular origin of this unexpected intracellular localization is solely dependent on the N-terminal part of the Gag protein, which lacks a functional sequence for myristoylation and plasma membrane targeting: replacement of the N-terminal domain of the MusD matrix protein by that of its closest relative—the Mason-Pfizer monkey virus—led to targeting of the MusD Gag to the plasma membrane, with viral particles budding and being released into the cell supernatant. These particles can further be pseudotyped with a heterologous envelope protein and become infectious, thus “reconstituting” a functional retrovirus prone to proviral insertions. Consistent with its retroviral origin, a sequence with a constitutive transport element-like activity can further be identified at the MusD 3′ untranslated region. A molecular scenario is proposed that accounts for the transition, during evolution, from an ancestral infectious betaretrovirus to the strictly intracellular MusD retrotransposon, involving not only the loss of the env gene but also an inability to escape the cell—via altered targeting of the Gag protein—resulting de facto in the generation of a very successful “intracellularized” insertional mutagen.
Transposable elements are major components of most eukaryotic genomes. Such sequences are generally defective for transposition and have little or no coding capacity. Because transposition can be highly mutagenic, mobile elements that remain functional are tightly repressed in all living species. Drosophila pericentromeric heterochromatin naturally contains transposition-defective, non-coding derivatives of a LINE retrotransposon related to the I-factor. The I-factor is a good model to study the regulation of transposition in vivo because, under specific conditions, current functional copies of this mobile element can transpose at high frequency, specifically in female germ cells, with deleterious effects including female sterility. However, this high transpositional activity becomes spontaneously repressed upon ageing or heat treatment, by a maternally transmitted, transgenerational epigenetic mechanism of unknown nature. We have analyzed, by quantitative real time RT-PCR, the RNA profile of the transposition-defective I-related sequences, in the Drosophila ovary during ageing and upon heat treatment, and also in female somatic tissues and in males, which are not permissive for I-factor transposition. We found evidence for a role of transcripts from these ancestral remnants in the natural epigenetic protection of the Drosophila melanogaster genome against the deleterious effects of new invasions by functional I-factors. These results provide a molecular basis for a probably widespread natural protection against transposable elements by persisting vestiges of ancient invasions.
We demonstrated previously that the cytosine deaminase APOBEC3G inhibits retrotransposition of two active murine endogenous retroviruses, namely intracisternal A-particles (IAP) and MusD, in an ex vivo assay where retrotransposition was monitored by selection of neo-marked elements. Sequencing of the transposed copies further disclosed extensive editing, resulting in a high load of G-to-A mutations. Here, we asked whether this G-to-A editing was associated with an impact of APOBEC3G on viral cDNA yields. To this end, we used a specially designed quantitative PCR method to selectively measure the copy number of transposed retroelements, in the absence of G418 selection. We show that human APOBEC3G severely reduces the number of MusD and IAP transposed cDNA copies, with no effect on the level of the intermediate RNA transcripts. The magnitude of the decrease closely parallels that observed when transposed copies are assayed by selection of G418-resistant cells. Moreover, sequencing of transposed elements recovered by PCR without prior selection of the cells reveals high-level editing. Using this direct method with a series of cytosine deaminases, we further demonstrate a similar dual effect of African green monkey APOBE3G, human APOBEC3F and murine APOBEC3 on MusD retrotransposition, with a distinct extent and site specificity for each editing activity. Altogether the data demonstrate that cytosine deaminases have a protective effect against endogenous retroviruses both by reducing viral cDNA levels and by introducing mutations in the transposed copies, thus inactivating them for subsequent rounds of retrotransposition. This dual, two-step effect likely participates in the efficient defense of the cell genome against invading endogenous retroelements.
Genome-wide screening of sequence databases for human endogenous retroviruses (HERVs) has led to the identification of 18 coding env genes, among which two—the syncytin genes—encode fusogenic ENV proteins possibly involved in placenta physiology. Here we show that a third ENV, originating from the most “recent” HERV-K(HML2) family, is functional. Immunofluorescence analysis of env-transduced cells demonstrates expression of the protein at the cell surface, and we show that the protein confers infectivity to simian immunodeficiency virus pseudotypes. Western blot analysis of the pseudotyped virions further discloses the expected specific cleavage of the ENV precursor protein. This functional ENV could play a role in the amplification—via infection of the germ line—of the HERV-K genomic copies, all the more as coding HERV-K gag and pol genes can similarly be found in the human genome, which could therefore generate infectious virions of a fully endogenous origin.
The human genome carries a high load of proviral-like sequences, called Human Endogenous Retroviruses (HERVs), which are the genomic traces of ancient infections by active retroviruses. These elements are in most cases defective, but open reading frames can still be found for the retroviral envelope gene, with sixteen such genes identified so far. Several of them are conserved during primate evolution, having possibly been co-opted by their host for a physiological role.
To characterize further their status, we presently sequenced 12 of these genes from a panel of 91 Caucasian individuals. Genomic analyses reveal strong sequence conservation (only two non synonymous Single Nucleotide Polymorphisms [SNPs]) for the two HERV-W and HERV-FRD envelope genes, i.e. for the two genes specifically expressed in the placenta and possibly involved in syncytiotrophoblast formation. We further show – using an ex vivo fusion assay for each allelic form – that none of these SNPs impairs the fusogenic function. The other envelope proteins disclose variable polymorphisms, with the occurrence of a stop codon and/or frameshift for most – but not all – of them. Moreover, the sequence conservation analysis of the orthologous genes that can be found in primates shows that three env genes have been maintained in a fully coding state throughout evolution including envW and envFRD.
Altogether, the present study strongly suggests that some but not all envelope encoding sequences are bona fide genes. It also provides new tools to elucidate the possible role of endogenous envelope proteins as susceptibility factors in a number of pathologies where HERVs have been suspected to be involved.
A recent in silico search for coding sequences of retroviral origin present in the human genome has unraveled two new envelope genes that add to the 16 genes previously identified. A systematic search among the latter for a fusogenic activity had led to the identification of two bona fide genes, named syncytin-1 and syncytin-2, most probably co-opted by primate genomes for a placental function related to the formation of the syncytiotrophoblast by cell-cell fusion. Here, we show that one of the newly identified envelope gene, named envP(b), is fusogenic in an ex vivo assay, but that its expression – as quantified by real-time RT-PCR on a large panel of human tissues – is ubiquitous, albeit with a rather low value in most tissues. Conversely, the second envelope gene, named envV, discloses a placenta-specific expression, but is not fusogenic in any of the cells tested. Altogether, these results suggest that at least one of these env genes may play a role in placentation, but most probably through a process different from that of the two previously identified syncytins.
A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians—from New World monkeys to humans—are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation—located in the TM subunit—was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution.
Sequences of retroviral origin occupy approximately 8% of the human genome. Most of these “retroviral” genes have lost their coding capacities since their entry into our ancestral genome millions of years ago, but some reading frames have remained open, suggesting positive selection. The complete sequencing of the human genome allowed a systematic search for retroviral envelope genes containing an open reading frame and resulted in the identification of 16 genes that we have characterized. We further showed, by quantitative reverse transcriptase PCR using specifically devised primers which discriminate between coding and noncoding elements, that all 16 genes are expressed in at least some healthy human tissues, albeit at highly different levels. All envelope genes disclose significant expression in the testis, three of them have a very high level of expression in the placenta, and a fourth is expressed in the thyroid. Besides their primary role as key molecules for viral entry, the envelope genes of retroviruses can induce cell-cell fusion, elicit immunosuppressive effects, and even protect against infection, and as such, endogenous retroviral envelope proteins have been tentatively identified in several reports as being involved in both normal and pathological processes. The present study provides a comprehensive survey of candidate genes and tools for a precise evaluation of their involvement in these processes.
We devised an indicator gene for retrotransposition based on an autocatalytic ribozyme element—the Tetrahymena thermophila 23S rRNA group I intron—which can self-splice in vitro and does not require—at variance with nuclear mRNA introns—any specific pathway and cellular component for the completion of the splicing process. Several constructs, with the Tetrahymena intron adequately modified so as to be inserted at various positions within a neomycin-containing cassette under conditions that restore the neomycin-coding sequence after splicing out of the intron, were assayed for splicing efficiency in mammalian cells in culture. We show, both by northern blot analysis and by the recovery of neomycin activity upon retroviral transduction of the cassettes, that splicing efficiency depends on both the local base pairing and the global position of the intron within the neomycin transcript, and that some constructs are functional. We further show that they allow the efficient sorting out of retrotransposition events when assayed, as a control, with a human LINE retrotransposon. These indicator genes should be of great help in elucidating the mechanisms of transposition of a series of retroelements associated with transcripts not prone to nuclear mRNA intron splicing and previously not opened to any retrotransposition assay.