Thierry Heidmann

LINE-mediated retrotransposition of marked Alu sequences

Alu elements are the most successful transposons in humans. They are 300-bp non-coding sequences transcribed by RNA polymerase III (Pol III) and are expected to retrotranspose with the aid of reverse transcriptases of cellular origin. We previously showed that human LINEs can generate cDNA copies of any mRNA transcript by means of a retroposition process involving reverse transcription and integration by the LINE-encoded endonuclease and reverse transcriptase. Here we show mobility of marked Alu sequences in human HeLa cells with the canonical features of a retrotransposition process, including splicing out of an autocatalytic intron introduced into the marked sequence, target site duplications of varying lengths and integrations into consensus A-rich sequences. We further show that the poly-A stretch at the Alu 3′ end is essential for mobility, that LINEs are required for transposition and that the rate of retroposition is 100–1,000 times higher for Alu transcripts than for control mRNAs, thus accounting for the high mutational activity of these elements observed in humans.

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.

Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution

Screening human sequence databases for endogenous retroviral elements with coding envelope genes has revealed 16 candidate genes that we assayed for their fusogenic properties. All 16 genes were cloned in a eukaryotic expression vector and assayed for cell–cell fusion by using a large panel of mammalian cells in transient transfection assays. Fusion was observed for two human endogenous retrovirus (HERV) envelopes, the previously characterized HERV-W envelope, also called syncytin, and a previously uncharacterized gene from the HERV-FRD family. Cells prone to env-mediated fusion were different for the two envelopes, indicating different receptor usage. A search for the FRDenv gene in primates indicated that the corresponding proviral element is present in all simians, from New World monkeys to humans, being absent only in prosimians. Cloning of the corresponding env genes in simians disclosed conservation of the fully coding status of the gene, and most remarkably, conservation of its fusogenic property. Finally, a Northern blot analysis for the expression of the FRD family among a series of human tissues demonstrated specific expression in the placenta, as previously demonstrated for the other fusogenic human envelope of the HERV-W family. Altogether, the present data have identified a previously uncharacterized envelope (that we propose to name syncytin 2 after renaming syncytin as syncytin 1) with a potential role in placenta formation, and the identification of the complete set of retroviral envelopes with fusogenic properties now allows a definite analysis of the possible role of HERV in this physiological process, via classical genetic approaches.

APOBEC3G cytidine deaminase inhibits retrotransposition of endogenous retroviruses

Endogenous retroviruses are multicopy retroelements accounting for nearly 10% of murine or human genomes. These retroelements spread into our ancestral genome millions of years ago and have acted as a driving force for genome evolution. Endogenous retroviruses may also be deleterious for their host, and have been implicated in cancers and autoimmune diseases. Most retroelements have lost replication competence because of the accumulation of inactivating mutations, but several, including some murine intracisternal A-particle (IAP) and MusD sequences, are still mobile. These elements encode a reverse transcriptase activity and move by retrotransposition, an intracellular copy-and-paste process involving an RNA intermediate. The host has developed mechanisms to silence their expression, mainly cosuppression and gene methylation. Here we identify another level of antiviral control, mediated by APOBEC3G, a member of the cytidine deaminase family that was previously shown to block HIV replication. We show that APOBEC3G markedly inhibits retrotransposition of IAP and MusD elements, and induces G-to-A hypermutations in their DNA copies. APOBEC3G, by editing viral genetic material, provides an ancestral wide cellular defence against endogenous and exogenous invaders.

High-molecular-mass APOBEC3G complexes restrict Alu retrotransposition

APOBEC3G (A3G) and related deoxycytidine deaminases are potent intrinsic antiretroviral factors. A3G is expressed either as an enzymatically active low-molecular-mass (LMM) form or as an enzymatically inactive high-molecular-mass (HMM) ribonucleoprotein complex. Resting CD4 T cells exclusively express LMM A3G, where it functions as a powerful postentry restriction factor for HIV-1. Activation of CD4 T cells promotes the recruitment of LMM A3G into 5- to 15-MDa HMM complexes whose function is unknown. Using tandem affinity purification techniques coupled with MS, we identified Staufen-containing RNA-transporting granules and Ro ribonucleoprotein complexes as specific components of HMM A3G complexes. Analysis of RNAs in these complexes revealed Alu and small Y RNAs, two of the most prominent nonautonomous mobile genetic elements in human cells. These retroelement RNAs are recruited into Staufen-containing RNA-transporting granules in the presence of A3G. Retrotransposition of Alu and hY RNAs depends on the reverse transcriptase machinery provided by long interspersed nucleotide elements 1 (L1). We now show that A3G greatly inhibits L1-dependent retrotransposition of marked Alu retroelements not by inhibiting L1 function but by sequestering Alu RNAs in cytoplasmic HMM A3G complexes away from the nuclear L1 enzymatic machinery. These findings identify nonautonomous Alu and hY retroelements as natural cellular targets of A3G and highlight how different forms of A3G uniquely protect cells from the threats posed by exogenous retroviruses (LMM A3G) and endogenous retroelements (HMM A3G).

Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene

In most mammalian species, a key process of placenta development is the fusion of trophoblast cells into a highly specialized, multinucleated syncytiotrophoblast layer, through which most of the maternofetal exchanges take place. Little is known about this process, despite the recent identification of 2 pairs of envelope genes of retroviral origin, independently acquired by the human (syncytin-1 and syncytin-2) and mouse (syncytin-A and syncytin-B) genomes, specifically expressed in the placenta, and with in vitro cell–cell fusion activity. By generating knockout mice, we show here that homozygous syncytin-A null mouse embryos die in utero between 11.5 and 13.5 days of gestation. Refined cellular and subcellular analyses of the syncytin-A-deficient placentae disclose specific disruption of the architecture of the syncytiotrophoblast-containing labyrinth, with the trophoblast cells failing to fuse into an interhemal syncytial layer. Lack of syncytin-A-mediated trophoblast cell fusion is associated with cell overexpansion at the expense of fetal blood vessel spaces and with apoptosis, adding to the observed maternofetal interface structural defects to provoke decreased vascularization, inhibition of placental transport, and fetal growth retardation, ultimately resulting in death of the embryo. These results demonstrate that syncytin-A is essential for trophoblast cell differentiation and syncytiotrophoblast morphogenesis during placenta development, and they provide evidence that genes captured from ancestral retroviruses have been pivotal in the acquisition of new, important functions in mammalian evolution.

Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins.

We have previously demonstrated that the envelope proteins of a murine and primate retrovirus are immunosuppressive in vivo. This property was manifested by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to have the env-expressing cells escape (at least transiently) immune rejection. Here, we analyzed the immunosuppressive activity of the human and murine syncytins. These are envelope genes from endogenous retroviruses independently coopted by ancestral hosts, conserved in evolution, specifically expressed in the placenta, and with a cell–cell fusogenic activity likely contributing to placenta morphogenesis. We show that in both humans and mice, one of the two syncytins (human syncytin-2 and mouse syncytin-B) is immunosuppressive and, rather unexpectedly, the other (human syncytin-1 and mouse syncytin-A) is not (albeit able to induce cell–cell fusion). Delineation of the immunosuppressive domain by deletion analysis, combined with a comparison between immunosuppressive and nonimmunosuppressive sequences, allowed us to derive a mutation rule targeted to specific amino acids, resulting in selective switch from immunosuppressive to nonimmunosuppressive envelope proteins and vice versa. These results unravel a critical function of retroviral envelopes, not necessarily “individually” selected for in the retrovirus endogenization process, albeit “tandemly” conserved in evolution for the syncytin pairs in primates and Muridae. Selective inactivation of immunosuppression, under conditions not affecting fusogenicity, should be important for understanding the role of this function in placental physiology and maternofetal tolerance.

Survey of human genes of retroviral origin: identification and transcriptome of the genes with coding capacity for complete envelope proteins.

ABSTRACT 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.

Paleovirology of ‘syncytins’, retroviral env genes exapted for a role in placentation

The development of the emerging field of ‘paleovirology’ allows biologists to reconstruct the evolutionary history of fossil endogenous retroviral sequences integrated within the genome of living organisms and has led to the retrieval of conserved, ancient retroviral genes ‘exapted’ by ancestral hosts to fulfil essential physiological roles, syncytin genes being undoubtedly among the most remarkable examples of such a phenomenon. Indeed, syncytins are ‘new’ genes encoding proteins derived from the envelope protein of endogenous retroviral elements that have been captured and domesticated on multiple occasions and independently in diverse mammalian species, through a process of convergent evolution. Knockout of syncytin genes in mice provided evidence for their absolute requirement for placenta development and embryo survival, via formation by cell–cell fusion of syncytial cell layers at the fetal–maternal interface. These genes of exogenous origin, acquired ‘by chance’ and yet still ‘necessary’ to carry out a basic function in placental mammals, may have been pivotal in the emergence of mammalian ancestors with a placenta from egg-laying animals via the capture of a founding retroviral env gene, subsequently replaced in the diverse mammalian lineages by new env-derived syncytin genes, each providing its host with a positive selective advantage.

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.