From ancestral infectious retroviruses to bona fide cellular genes: role of the captured syncytins in placentation.

During their replication, infectious retroviruses insert a reverse-transcribed cDNA copy of their genome, a "provirus", into the genome of their host. If the infected cell belongs to the germline, the integrated provirus can become "fixed" within the host genome as an endogenous retrovirus and be transmitted vertically to the progeny in a Mendelian fashion. Based on the numerous proviral sequences that are recovered within the genomic DNA of vertebrates--up to ten percent in the case of mammals--such events must have occurred repeatedly during the course of millions of years of evolution. Although most of the ancient proviral sequences have been disrupted, a few "endogenized" retroviral genes are conserved and still encode functional proteins. In this review, we focus on the recent discovery of genes derived from the envelope glycoprotein-encoding (env) genes of endogenous retroviruses that have been domesticated by mammals to carry out an essential function in placental development. They were called syncytins based on the membrane fusogenic capacity that they have kept from their parental env gene and which contributes to the formation of the placental fused cell layer called the syncytiotrophoblast, at the materno-fetal interface. Remarkably, the capture of syncytin or syncytin-like genes, sometimes as pairs, was found to have occurred independently from different endogenous retroviruses in diverse mammalian lineages such as primates--including humans--, muroids, leporids, carnivores, caviids, and ovis, between around 10 and 85 million years ago. Knocking out one or both mouse syncytin-A and -B genes provided evidence that they indeed play a critical role in placentation. We discuss the possibility that the immunosuppressive domain embedded within retroviral envelope glycoproteins and conserved in syncytin proteins, may be involved in the tolerance of the fetus by the maternal immune system. Finally, we speculate that the capture of a founding syncytin-like gene could have been instrumental in the dramatic transition from egg-laying to placental mammals.

A syncytin-like endogenous retrovirus envelope gene of the guinea pig specifically expressed in the placenta junctional zone and conserved in Caviomorpha.

Syncytins are genes of retroviral origin that have been co-opted by mammalian hosts for a function in placentation. Two such genes have already been identified in simians, as well as two distinct, unrelated ones in Muridae and a fifth in the rabbit. Here we searched for similar genes in the guinea pig, which belongs to the Caviomorpha lineage within the Hystricognathi suborder of rodents and displays a placental structural organization with several characteristic features comparable to those of the human organ, including deep trophoblast invasion of maternal tissues. An in silico search for envelope (env) genes with full coding capacity identified a candidate gene that showed specific expression in the placenta, as revealed by RT-qPCR using RNAs from a large panel of tissues. This gene belongs to an endogenous retroviral element present at a single-copy in the guinea pig genome, still displaying a retroviral organization - with a degenerate gag and pol, but an intact env gene. In situ hybridization of guinea pig placenta sections demonstrated specific expression at the level of the invasive trophoblast-containing junctional zone, as observed in humans for syncytin-1 and consistent with a role in invasion of the maternal uterine tissues. The identified gene displays a conserved open reading frame in the Caviomorpha, consistent with an entry date >30 million years, and sequence analyses showed purifying selection of the gene. Conclusively, despite the absence of a demonstrated fusogenic activity, it is likely that the identified env gene - that we named syncytin-like env-Cav1 - exerts a physiological function possibly related to trophoblast invasion, in the course of caviomorph placentation.

Expression of the fusogenic HERV-FRD Env glycoprotein (syncytin 2) in human placenta is restricted to villous cytotrophoblastic cells.

Recently, the expression of a human endogenous retrovirus HERV-FRD, able to encode a fusogenic envelope protein (syncytin 2), has been observed in human placenta. The aim of the present study was to localize the expression of syncytin 2 in first trimester placenta. In addition, we investigated the presence of HERV-FRD transcripts during the in vitro differentiation of isolated villous and extravillous trophoblastic cells from first trimester chorionic villi. Using a monoclonal antibody specifically raised against the HERV-FRD Env protein, syncytin 2 was immunolocalized only in the villous trophoblast of the chorionic villi, at the level of cytotrophoblastic cells. Interestingly, immunostaining was not observed in all cells but only in some of them, and was detected, more frequently, at the membrane level at the interface between the cytotrophoblastic cells and syncytiotrophoblast. Labeling was observed neither in the syncytiotrophoblast nor in the mesenchymal core of the villi nor in the extravillous trophoblast. In vitro detection of HERV-FRD transcripts was restricted to villous trophoblastic cells and decreased significantly with time in culture. These results suggest that syncytin 2 might play a role in human trophoblastic cell fusion.

LINEs, SINEs and processed pseudogenes: parasitic strategies for genome modeling.

Two major classes of retrotransposons have invaded eukaryotic genomes: the LTR retrotransposons closely resembling the proviral integrated form of infectious retroviruses, and the non-LTR retrotransposons including the widespread, autonomous LINE elements. Here, we review the modeling effects of the latter class of elements, which are the most active in humans, and whose enzymatic machinery is subverted to generate a large series of "secondary" retroelements. These include the processed pseudogenes, naturally present in all eukaryotic genomes possessing non-LTR retroelements, and the very successful SINE elements such as the human Alu sequences which have evolved refined parasitic strategies to efficiently bypass the original "protectionist" cis-preference of LINEs for their own retrotransposition.

Human endogenous retroviruses: from infectious elements to human genes.

Mammalian genomes contain a heavy load (42% in humans) of retroelements, which are mobile sequences requiring reverse transcription for their replicative transposition. A significant proportion of these elements is of retroviral origin, with thousands of sequences resembling the integrated form of infectious retroviruses, with two LTRs bordering internal regions homologous to the gag, prt, pol, and env genes. These elements, named endogenous retroviruses (ERVs), are most probably the proviral remnants of ancestral germ-line infections by active retroviruses, which have thereafter been transmitted in a Mendelian manner. The complete sequencing of the human genome now allows a comprehensive survey of human ERVs (HERVs), which can be grouped according to sequence homologies into approximately 80 distinct families, each containing a few to several hundred elements. As reviewed here, strong similarities between HERVs and present-day retroviruses can be inferred from phylogenetic analyses on the reverse transcriptase (RT) domain of the pol gene or the transmembrane subunit (TM) of the env gene, which disclose interspersion of both classes of elements and suggest a common history and shared ancestors. Similarities are also observed at the functional levels, since despite the fact that most HERVs have accumulated mutations, deletions, and/or truncations, several elements still possess some of the functions of retroviruses, with evidence for viral-like particle formation, and occurrence of envelope proteins allowing cell-cell fusion and even conferring infectivity to pseudotypes. Along this line, a genomewide screening for human retroviral genes with coding capacity has revealed 16 fully coding envelope genes. These genes are transcribed in several healthy tissues including the placenta, three of them at a very high level. Besides their impact in modelling the genome, HERVs thus appear to contain still active genes, which most probably have been subverted by the host for its benefit and should be considered as bona fide human genes. Some of their characteristic features and possible physiological roles, as well as potential pathological effects inherited from their retroviral ancestors are also reviewed.

Identification of four families of yCCR4- and Mg2+-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding.

BACKGROUND: The yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR) motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg2+-dependent endonuclease core. RESULTS: Here, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals), which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner. CONCLUSIONS: We have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals.

Identification, phylogeny, and evolution of retroviral elements based on their envelope genes.

Phylogenetic analyses of retroviral elements, including endogenous retroviruses, have relied essentially on the retroviral pol gene expressing the highly conserved reverse transcriptase. This enzyme is essential for the life cycle of all retroid elements, but other genes are also endowed with conserved essential functions. Among them, the transmembrane (TM) subunit of the envelope gene is involved in virus entry through membrane fusion. It has also been reported to contain a domain, named the immunosuppressive domain, that has immunosuppressive properties most probably essential for virus spread within the host. This domain is conserved among a large series of retroviral elements, and we have therefore attempted to generate phylogenetic links between retroviral elements identified from databases following tentative alignments of the immunosuppressive domain and adjacent sequences. This allowed us to unravel a conserved organization among TM domains, also found in the Ebola and Marburg filoviruses, and to identify a large number of human endogenous retroviruses (HERVs) from sequence databases. The latter elements are part of previously identified families of HERVs, and some of them define new families. A general phylogenetic analysis based on the TM proteins of retroelements, and including those with no clearly identified immunosuppressive domain, could then be derived and compared with pol-based phylogenetic trees, providing a comprehensive survey of retroelements and definitive evidence for recombination events in the generation of both the endogenous and the present-day infectious retroviruses.

A truncated form of the human CAF-1 p150 subunit impairs the maintenance of transcriptional gene silencing in mammalian cells.

Chromatin assembly factor 1 (CAF-1) is a protein complex formed of three subunits, p150, p60, and p48, conserved from the yeast Saccharomyces cerevisiae to humans, which can promote nucleosome assembly onto newly replicated DNA. In S. cerevisiae, deletion of the genes encoding any of the three CAF-1 subunits (cacDelta mutants), although nonlethal, results in a silencing defect of genes packaged into heterochromatin. Here we report on a mammalian cell model that we devised to monitor gene silencing and its reversal in a quantitative manner. This model relies on the use of a cell line stably transfected with a reporter gene in a silenced state. Reversal of reporter gene silencing was achieved upon treatment of the cells with 5-azacytidine, which resulted in the demethylation of the reporter gene copies. We show that expression of a cDNA for the human p150 CAF-1 subunit harboring 5' truncations, but not that of a cDNA encoding the full-length p150 CAF-1 subunit, increases by more than 500-fold the frequency at which transcriptional silencing of the reporter gene copies is reversed in these cells. Reversal of gene silencing is dependent upon expression of a truncated protein, possibly acting as a dominant negative mutant of the wild-type CAF-1, is associated with alterations in chromatin structure as measured by an endonuclease sensitivity assay and is not associated with detectable changes in the methylation status of the silenced genes. These results suggest that the role of CAF-1 in the epigenetic control of gene expression has been conserved between yeast and mammals, despite the lack of DNA methylation in yeast chromatin.

Characterization of the three HERV-H proviruses with an open envelope reading frame encompassing the immunosuppressive domain and evolutionary history in primates.

The HERV-H family is one of the largest human endogenous retrovirus families, with approximately 1000 elements. Using a direct coupled in vitro transcription/translation approach (PTT for protein truncation test) and an extended series of primers on human genomic DNA, on monochromosomal hybrids and on a BAC library, we could demonstrate that there are only three envelopes with a large open reading frame encompassing the immunosuppressive (ISU) domain, corresponding to 62-, 60-, and 59-kDa potential translational products. The associated proviruses, HERV-H/env62, HERV-H/env60, and HERV-H/env59 were sequenced together with their flanking DNA and mapped by FISH, and their entry times within the primate lineage were determined. Analysis of the LTR sequences revealed numerous recombinational and/or homogenization events in the course of evolution, with divergences between 5' and 3' LTRs higher than expected for a simple time-dependent genetic drift. PTT analyses further revealed that the three large envelopes in humans are prematurely stopped in the majority of primates, and sequencing of the largest envelope gene, from HERV-H/env62, in five human individuals revealed two polymorphic sites. The results are consistent with the absence of a strong selective pressure for the conservation of a functional envelope gene of possible benefit for the host, but do not exclude somatic effects possibly associated with the immunosuppressive domain carried by these genes.

Human LINE retrotransposons generate processed pseudogenes.

Long interspersed elements (LINEs) are endogenous mobile genetic elements that have dispersed and accumulated in the genomes of higher eukaryotes via germline transposition, with up to 100,000 copies in mammalian genomes. In humans, LINEs are the major source of insertional mutagenesis, being involved in both germinal and somatic mutant phenotypes. Here we show that the human LINE retrotransposons, which transpose through the reverse transcription of their own transcript, can also mobilize transcribed DNA not associated with a LINE sequence by a process involving the diversion of the LINE enzymatic machinery by the corresponding mRNA transcripts. This results in the 'retroposition' of the transcribed gene and the formation of new copies that disclose features characteristic of the widespread and naturally occurring processed pseudogenes: loss of intron and promoter, acquisition of a poly(A) 3' end and presence of target-site duplications of varying length. We further show-by introducing deletions within either coding sequence of the human LINE-that both ORFs are necessary for the formation of the processed pseudogenes, and that retroviral-like elements are not able to produce similar structures in the same assay. Our results strengthen the unique versatility of LINEs as genome modellers.

Members of the SRY family regulate the human LINE retrotransposons.

LINEs are endogenous mobile genetic elements which have dispersed and accumulated in the genomes of most higher eukaryotes via germline transposition, with up to 100 000 copies for the human LINE-1 (L1H) sequences. Although severely repressed in most normal tissues, L1H is still functional, with evidence for both germline and somatic-essentially in tumors-transpositions. Yet, no transcription factor that could regulate their transcription and be responsible for their transposition has hitherto been described. Here we show that factors belonging to the family of the testis-determining factor gene SRY (the SOX family) can modulate L1H promoter activity over a 10-fold range in a transient transfection assay using a luciferase reporter gene. These effects depend on two functional SRY binding sites which can be identified within the L1H promoter via mobility shift assays. Induction of endogenous L1Hs upon ectopic expression of the SOX11 transcription factor is further demonstrated, thus strengthening the physiological relevance of these new-and highly dispersed-target sites for the otherwise unclassical transcription factors of the SRY family.

Cosuppression of I transposon activity in Drosophila by I-containing sense and antisense transgenes.

We have previously shown that the activity of functional I elements introduced into Drosophila devoid of such elements can be repressed by transgenes containing an internal nontranslatable part of the I element itself and that this repressing effect presents features characteristic of homology-dependent gene silencing or cosuppression. Here we show that transgenes containing a fragment of the I element in antisense orientation induce I-element silencing with the same characteristic features as the corresponding sense construct: namely, repression takes several generations to be fully established, with similar rates for sense and antisense constructs, and it is only maternally transmitted, with reversal of the effect through paternal transmission. We also show that transcription of the transgenes is necessary to produce the silencing effect and that repression can be maintained for at least one generation following elimination of the transgenes, thus strongly suggesting that a transgene product and not the transgene per se is the essential intermediate in the silencing effect. The data presented strongly support models in which the repressing effect of antisense transcripts involves the same mechanisms as cosuppression by sense constructs and emphasize the role of symmetrically acting nucleic acid structures in mediating repression.

Characterization of a mammalian gene related to the yeast CCR4 general transcription factor and revealed by transposon insertion.

Murine intracisternal A-particles (IAPs) are reiterated retrovirus-like transposable elements that can act as insertional mutagens. Accordingly, we previously identified a chimeric transcript initiated at an IAP promoter and extending through a 3'-located open reading frame with significant similarity to the C-terminal domain of the yeast CCR4 general transcription factor. In this report, we characterize the corresponding murine gene, mCCR4, and its human homologue, thus providing the first description of CCR4-like factors in mammals. cDNA cloning revealed two mCCR4 mRNAs of 2.7 and 3.1 kilobases, differing by their transcription start sites within the native mCCR4 gene promoter, and encoding a putative 430-amino acid protein. The mCCR4 gene contains three exons and two introns spanning almost 27 kilobases. The IAP insertion, detected only in some laboratory mouse strains, is recent and lies within the first intron. The 5'-region of the gene has features of housekeeping gene promoters. It lacks a TATA box but contains a CpG island and Sp1 sites. This region discloses strong promoter activity in transient transfection assays and also stimulates transcription in the reverse orientation, a feature common to other CpG island-containing promoters. Transcripts were detected in all the organs tested, although at a variable level, and displayed no strain-dependent differences relative to the IAP insertion, suggesting the existence of mechanisms preserving mCCR4 transcription from the usually deleterious effects of intronic transposition. The strong amino acid conservation between the human, murine, and the previously identified Xenopus CCR4-like proteins, is consistent with an important and conserved role for this protein in vertebrates.

ERV-3 envelope expression and congenital heart block: what does a physiological knockout teach us.

Congenital heart block is a serious condition with significant mortality due in most cases to the transplacental transfer of autoantibodies from an otherwise asymptomatic mother. Although SSA/Ro and SSB/La autoantibodies have been implicated, attention has focused recently on autoantibodies to envelope proteins of endogenous retrovirus-3 (ERV-3). We have recently identified in 1% of the caucasian population a natural knock out of ERV-3 due to a premature stop mutation generating a severely truncated form of the protein [corrected]. If a pregnant female homozygous for the truncated form of the ERV-3 carries a foetus expressing the entire protein, the mother might be expected to acquire high titre immunity, while the foetus homozygous for the truncated form would not be expected to immunise its mother. In order to test whether this naturally occurring model could shed light on the pathogenesis of CHB, we determined the status of the ERV-3 stop polymorphism in 12 mothers of CHB infants [corrected]. The fact that none was homozygous for the stop mutation tends to rule out a role for the stop polymorphism of the mothers in the generation of the CHB disease, but does not exclude that other polymorphisms might be involved [corrected].

The long terminal repeats of the HERV-H human endogenous retrovirus contain binding sites for transcriptional regulation by the Myb protein.

Binding sites for transcriptional regulation by the Myb protein have been identified in the long terminal repeats (LTRs) of the H-type human endogenous retrovirus-like elements (HERV-H). Transfection assays using reporter plasmids containing the luciferase gene under the control of a HERV-H LTR disclosed a sevenfold increase in promoter activity in human teratocarcinoma cells when cotransfected with an expression vector for the Myb protein. Binding sites for Myb were unambiguously identified within the LTR by both DNase I footprinting experiments and mobility shift assays using a bacterially expressed purified Myb recombinant protein. Possible roles of these Myb-responsive elements are discussed.

ERV-L elements: a family of endogenous retrovirus-like elements active throughout the evolution of mammals.

We have previously identified in the human genome a family of 200 endogenous retrovirus-like elements, the HERV-L elements, disclosing similarities with the foamy retroviruses and which might be the evolutionary intermediate between classical intracellular retrotransposons and infectious retroviruses. Southern blot analysis of a large series of mammalian genomic DNAs shows that HERV-L-related elements-so-called ERV-L-are present among all placental mammals, suggesting that ERV-L elements were already present at least 70 million years ago. Most species exhibit a low copy number of ERV-L elements (from 10 to 30), while simians (not prosimians) and mice (not rats) have been subjected to bursts resulting in increases in the number of copies up to 200. The burst of copy number in primates can be dated to shortly after the prosimian and simian branchpoint, 45 to 65 million years ago, whereas murine species have been subjected to two much more recent bursts (less than 10 million years ago), occurring after the Mus/Rattus split. We have amplified and sequenced 360-bp ERV-L internal fragments of the highly conserved pol gene from a series of 22 mammalian species. These sequences exhibit high percentages of identity (57 to 99%) with the murine fully coding MuERV-L element. Phylogenetic analyses allowed the establishment of a plausible evolutionary scheme for ERV-L elements, which accounts for the high level of sequence conservation and the widespread dispersion among mammals.

Taming of transposable elements by homology-dependent gene silencing.

Transposable elements can invade virgin genomes within a few generations, after which the elements are 'tamed' and retain only limited transpositional activity. Introduction of the I element, a transposon similar to mammalian LINE elements, into Drosophila melanogaster genomes devoid of such elements initially results in high-frequency transposition of the incoming transposon, high mutation rate, chromosomal nondisjunction and female sterility, a syndrome referred to as hybrid dysgenesis (for review, see refs 2-4); a related syndrome has also been described in mammals. High-frequency transposition is transient, as the number of I elements reaches a finite value and transposition ceases after approximately ten generations. It has been proposed that the I elements encode a factor that negatively regulates their own transcription, but evidence for such a mechanism is lacking. Using the hybrid dysgenesis syndrome in Drosophila as a model, we show here that transpositional activity of the I element can be repressed by prior introduction of transgenes expressing a small internal region of the I element. This autoregulation presents features characteristic of homology-dependent gene silencing, a process known as cosuppression. Repression does not require any translatable sequence, its severity correlates with transgene copy number and it develops in a generation-dependent manner via germline transmission of a silencing effector in females only. These results demonstrate that transposable elements are prone to and can be tamed by homology-dependent gene silencing, a process that may have emerged during the course of evolution as a specific defense mechanism against these elements.

Tumor cells expressing a retroviral envelope escape immune rejection in vivo.

A model system for the in vivo control of tumor cell proliferation by the immune system has been used to assay for the possible immunosuppressive activity of retroviral proteins. Expression vectors for the entire or the transmembrane subunit of the Moloney murine leukemia virus envelope protein were constructed, as well as control vectors for irrelevant transmembrane proteins-or no protein. They were introduced either into MCA205 murine tumor cells, which do not proliferate upon s.c. injection into an allogeneic host, or into CL8.1 murine tumor cells, which overexpress class I antigens and are rejected in a syngeneic host. In both cases, expression of the complete envelope protein or of the transmembrane subunit resulted in tumor growth in vivo, with no effect of control vectors. Tumor cell growth results from inhibition of the host immune response, as the envelope-dependent effect was no more observed for MCA205 cells in syngeneic mice or for CL8.1 cells in x-irradiated mice. This inhibition is local because it is not observed at the level of control tumor cells injected contralaterally. These results suggest a noncanonical function of retroviral envelopes in the "penetrance" of viral infections, as well as a possible involvement of the envelope proteins of endogenous retroviruses in tumoral processes.

Physiological knockout of the envelope gene of the single-copy ERV-3 human endogenous retrovirus in a fraction of the Caucasian population.

ERV-3 is an evolutionarily conserved single-copy human endogenous retrovirus with a coding envelope gene potentially involved in important placental functions. We have investigated the sequence variability of this gene among 150 unrelated Caucasian individuals and found eight polymorphic sites. One of them corresponds to the introduction of a stop codon resulting in the production of a severely truncated ERV-3 envelope protein lacking both the fusion peptide and the immunosuppressive domain of the protein. The stop codon is observed in a homozygous state in approximately 1% of Caucasian individuals without evidence for counterselection, thus precluding the involvement of any essential function of the gene in placental implantation and development. This natural knockout provides a mean to investigate other potential roles for this otherwise highly conserved gene.

A nuclear matrix/scaffold attachment region co-localizes with the gypsy retrotransposon insulator sequence.

The 5'-untranslated region of the Drosophila gypsy retrotransposon contains an "insulator," which disrupts the interactions between enhancer and promoter elements located apart. The insulator effect is dependent on the suppressor of Hairy-wing (su(Hw)) protein, which binds to reiterated sites within the 350 base pairs of the gypsy insulator, whereby it additionally acts as a transcriptional activator of gypsy. Here, we show that the 350-base pair su(Hw) binding site-containing gypsy insulator behaves in addition as a matrix/scaffold attachment region (MAR/SAR), involved in interactions with the nuclear matrix. In vitro experiments using nuclear matrices from Drosophila, murine, and human cells demonstrate specific binding of the gypsy insulator, not observed with any other sequence within the retrotransposon. Moreover, we show that the gypsy insulator, like previously characterized MAR/SARs, specifically interacts with topoisomerase II and histone H1, i.e. with two essential components of the nuclear matrix. Finally, experiments within cells in culture demonstrate differential effects of the gypsy MAR sequence on reporter genes, namely no effect under conditions of transient transfection and a repressing effect in stable transformants, as expected for a sequence involved in chromatin structure and organization. A model for the gypsy insulator, which combines within a short "compacted" retroviral sequence three functional domains (insulator, enhancer, and the presently unraveled MAR/SAR) dispersed within more extended regions in other "boundary" domains, is discussed in relation to previously proposed models for insulation.