Biomedical subjects

Thierry Heidmann

Publications and source records attributed to Thierry Heidmann.

At least 19 recordsLinked to original sources

PubMed2006

Murine MusD retrotransposon: structure and molecular evolution of an "intracellularized" retrovirus.

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.

3' Untranslated Regions↗

PubMed2006

Identification of an infectious progenitor for the multiple-copy HERV-K human endogenous retroelements.

Human Endogenous Retroviruses are expected to be the remnants of ancestral infections of primates by active retroviruses that have thereafter been transmitted in a Mendelian fashion. Here, we derived in silico the sequence of the putative ancestral "progenitor" element of one of the most recently amplified family - the HERV-K family - and constructed it. This element, Phoenix, produces viral particles that disclose all of the structural and functional properties of a bona-fide retrovirus, can infect mammalian, including human, cells, and integrate with the exact signature of the presently found endogenous HERV-K progeny. We also show that this element amplifies via an extracellular pathway involving reinfection, at variance with the non-LTR-retrotransposons (LINEs, SINEs) or LTR-retrotransposons, thus recapitulating ex vivo the molecular events responsible for its dissemination in the host genomes. We also show that in vitro recombinations among present-day human HERV-K (also known as ERVK) loci can similarly generate functional HERV-K elements, indicating that human cells still have the potential to produce infectious retroviruses.

Amino Acid Sequence↗

PubMed2006

Mobility and integration sites of a murine C57BL/6 melanoma endogenous retrovirus involved in tumor progression in vivo.

Tumor development is a multistep process in which both genetic and epigenetic events cooperate for the emergence of a malignant clone with metastatic properties. The possibility that endogenous retroviruses promote the expansion of a neoplastic clone by subverting immunosurveillance has been proposed and recently demonstrated in the case of the B16 murine melanoma, which spontaneously express the melanoma-associated retrovirus (MelARV). Indeed, knocking down, by RNA interference, this endogenous retrovirus resulted in the rejection of the tumor cells in immunocompetent mice, without any alteration of their transformed phenotype. Here, we characterize the MelARV proviruses present in the B16 melanoma. Complete sequencing of the viral genomic RNA and characterization of the integration sites within both the B16 tumor cells and a subline selected in vivo for increased metastatic activity disclosed mobility of the element with new proviral insertions targeting critical genes and altering their transcriptional profile. The results show that MelARV can act both at the genetic level, inducing mutations by insertion, and at the epigenetic level, promoting immunosuppression of the host. These properties may as well be relevant to human tumors, such as germline tumors and melanoma, where endogenous retroviruses are active.

Adaptor Proteins, Signal Transducing↗

PubMed2006

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).

APOBEC-3G Deaminase↗

PubMed2006

A recombinant endogenous retrovirus amplified in a mouse neuroblastoma is involved in tumor growth in vivo.

The theory of immunoediting postulates that tumor cells exhibit a reduced immunogenicity to escape eradication by the host immune system. It has been proposed that endogenous retroviruses--provided that they are active--could play a role in this process, via the immunosuppressive domain carried by their envelope protein. Here, we demonstrate that the Neuro-2a tumor cell line--originating from a spontaneous A/J mouse neuroblastoma--produces an infectious retrovirus that most probably results from a recombination event between 2 mouse endogenous retroviral elements. This Neuro-2a-associated recombinant retrovirus derives from the unique ecotropic provirus located at the Emv-1 locus, but with a gag sequence conferring B-tropism, thus allowing its high-level amplification in Neuro-2a cells. We show that knocking down -by RNA interference- this endogenous retrovirus in Neuro-2a cells has no effect on the transformed phenotype of the cells, but results in delayed tumor growth and prolonged animal survival, following engraftment of the cells into immunocompetent mice. Recombination between endogenous retroviruses, amplification of the resulting element and high-level expression of its immunosuppressive activity are therefore likely steps of an immunoediting process, leading to an invading tumor.

Amino Acid Sequence↗

PubMed2006

Dual inhibitory effects of APOBEC family proteins on retrotransposition of mammalian endogenous retroviruses.

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.

APOBEC-3G Deaminase↗

PubMed2005

Crystal structure of a pivotal domain of human syncytin-2, a 40 million years old endogenous retrovirus fusogenic envelope gene captured by primates.

HERV-FRD is a human endogenous retrovirus that entered the human genome 40 million years ago. Its envelope gene, syncytin-2, was diverted by an ancestral host most probably because of its fusogenic property, for a role in placenta morphogenesis. It was maintained in a functional state in all primate branches as a bona fide cellular gene, submitted to a very low mutation rate as compared to infectious retrovirus genomes. The structure of the syncytin-2 protein thus provides a good insight into that of the oldest mammalian retroviral envelope. Here, we report the crystal structure of a central fragment of its "fossil" ectodomain, allowing a remarkable superposition with the structures of the corresponding domains of present-day infectious retroviruses, in spite of a more than 60% divergent sequence. These results suggest the existence of a unique structural solution selected by these proteins for their fusogenic function.

Amino Acid Sequence↗

PubMed2005

Comprehensive search for intra- and inter-specific sequence polymorphisms among coding envelope genes of retroviral origin found in the human genome: genes and pseudogenes.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Alleles↗

PubMed2005

L1-mediated retrotransposition of murine B1 and B2 SINEs recapitulated in cultured cells.

SINEs are short interspersed nucleotide elements with transpositional activity, present at a high copy number (up to a million) in mammalian genomes. They are 80-400 bp long, non-coding sequences which derive either from the 7SL RNA (e.g. human Alus, murine B1s) or tRNA (e.g. murine B2s) polymerase III-driven genes. We have previously demonstrated that Alus very efficiently divert the enzymatic machinery of the autonomous L1 LINE (long interspersed nucleotide element) retrotransposons to transpose at a high rate. Here we show, using an ex vivo assay for transposition, that both B1 and B2 SINEs can be mobilized by murine LINEs, with the hallmarks of a bona fide retrotransposition process, including target site duplications of varying lengths and integrations into A-rich sequences. Despite different phylogenetic origins, transposition of the tRNA-derived B2 sequences is as efficient as that of the human Alus, whereas that of B1s is 20-100-fold lower despite a similar high copy number of these elements in the mouse genome. We provide evidence, via an appropriate nucleotide substitution within the B1 sequence in a domain essential for its intracellular targeting, that the current B1 SINEs are not optimal for transposition, a feature most probably selected for the host sake in the course of evolution.

Animals↗

PubMed2005

Endogenous retrovirus expression is required for murine melanoma tumor growth in vivo.

Tumor development is a multistep process in which both genetic and epigenetic events cooperate for the emergence of a malignant clone. The possibility that endogenous retroviruses promote the expansion of a neoplastic clone by subverting immune surveillance has been proposed, but remained elusive. Here we show that knocking down-by RNA interference-an endogenous retrovirus spontaneously induced in the B16 murine melanoma results in the rejection of the tumor cells in immunocompetent mice, under conditions where control melanoma cells grow into lethal tumors. The knockdown does not modify the transformed phenotype of the cells, as measured both in vitro by a soft agar assay and in vivo by tumor cell proliferation in immunoincompetent (X-irradiated and severe combined immunodeficiency) mice. Tumor rejection can be reverted upon adoptive transfer of regulatory T cells from control melanoma-engrafted mice, as well as upon reexpression of the sole envelope gene of the endogenous retrovirus in the knocked down cells. These results show that endogenous retroviruses can be essential for a regulatory T-cell-mediated subversion of immune surveillance and could be relevant to human tumors where such elements-and especially their envelope gene-are induced.

Animals↗

PubMed2005

Functional characterization of two newly identified Human Endogenous Retrovirus coding envelope genes.

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.

Animals↗

PubMed2005

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.

APOBEC-3G Deaminase↗

PubMed2005

Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae.

Recently, we and others have identified two human endogenous retroviruses that entered the primate lineage 25-40 million years ago and that encode highly fusogenic retroviral envelope proteins (syncytin-1 and -2), possibly involved in the formation of the placenta syncytiotrophoblast layer generated by trophoblast cell fusion at the materno-fetal interface. A systematic in silico search throughout mouse genome databases presently identifies two fully coding envelope genes, present as unique copies and unrelated to any known murine endogenous retrovirus, that we named syncytin-A and -B. Quantitative RT-PCR demonstrates placenta-specific expression for both genes, with increasing transcript levels in this organ from 9.5 to 14.5 days postcoitum. In situ hybridization of placenta cryosections further localizes these transcripts in the syncytiotrophoblast-containing labyrinthine zona. Consistently, we show that both genes can trigger cell-cell fusion in ex vivo transfection assays, with distinct cell type specificities suggesting different receptor usage. Genes orthologous to syncytin-A and -B and disclosing a striking conservation of their coding status are found in all Muridae tested (mouse, rat, gerbil, vole, and hamster), dating their entry into the rodent lineage approximately 20 million years ago. Together, these data strongly argue for a critical role of syncytin-A and -B in murine syncytiotrophoblast formation, thus unraveling a rather unique situation where two pairs of endogenous retroviruses, independently acquired by the primate and rodent lineages, would have been positively selected for a convergent physiological role.

Animals↗

PubMed2005

Role of poly(A) tail length in Alu retrotransposition.

Alu are mobile noncoding Short INterspersed Elements (SINEs) present at a million copies in the human genome. Using marked Alu sequences in an ex vivo assay, we previously showed that they are mobilized through diversion of the LINE (Long INterspersed Elements) retrotransposition machinery, with the poly(A) tail of the Alu being required for their mobility. Here we show that other homopolymeric tracts cannot functionally replace the Alu poly(A) tail, and that the Alu transposition rate varies over a two-log range depending on the poly(A) tail length. Variation is according to a sigmoid-shaped curve with a lag observed for tails shorter than 15 nt and a plateau reached for tails longer than 50 nt, consistent with the binding of a limited number of a protein component requiring multiple contacts for a productive interaction with the poly(A) stretch. This analysis indicates that most of the naturally occurring genomic Alu, owing to their pA tail length, should be poor substrates for the LINE machinery, a feature possibly "selected" for the host sake.

Alu Elements↗

PubMed2005

Identification of a functional envelope protein from the HERV-K family of human endogenous retroviruses.

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.

Endogenous Retroviruses↗

PubMed2004

An active murine transposon family pair: retrotransposition of "master" MusD copies and ETn trans-mobilization.

The ETn (Early Transposon) elements are among the most active murine mobile sequences, being responsible for a series of mutations by insertion in vivo. Yet they are noncoding, and it had long been suspected that ETn are mobilized in trans by coding-competent elements, most probably from the closely related MusD family of LTR-retrotransposons. A genome-wide in silico search for coding-competent MusD elements identified a total of nine such copies, which we cloned and marked to test their transpositional activity, using an ex vivo assay in heterologous cells. Three copies were found to be autonomous for transposition, with each gag, pro, and pol MusD gene absolutely required for mobility. These active MusD copies specifically trigger retrotransposition of marked ETn elements with high efficiency, by complementation in trans. Characterization of the structures of de novo transposed MusD and ETn marked elements, as well as of their integration sites, disclosed canonical retroviral-like retrotransposition, with 6-bp target site duplications common to both elements. These results highlight the parasitic molecular strategies that are used by the ETn elements for their mobility, and unambiguously identify their "master genes."

Animals↗

PubMed2004

Identification of autonomous IAP LTR retrotransposons mobile in mammalian cells.

Mammalian genomes contain two main classes of retrotransposons, the well-characterized long and short interspersed nuclear elements, which account for approximately 30% of the genome, and the long terminal repeat (LTR) retrotransposons, which resemble the proviral integrated form of retroviruses, except for the absence of an envelope gene in some cases. Genetic studies confirmed mobility of the latter class of elements in mice, with a high proportion of phenotypic mutations consequent to transposition of the intracisternal A particle (IAP) family of LTR retrotransposons. Using the mouse genome sequence and an efficient ex vivo retrotransposition assay, we identified functional, master IAP copies that encode all the enzymatic and structural proteins necessary for their autonomous transposition in heterologous cells. By introducing mutations, we found that the three genes gag, prt and pol are all required for retrotransposition and identified the IAP gene products by electron microscopy in the form of intracellular A-type particles in the transfected cells. These prototypic elements, devoid of an envelope gene, are the first LTR retrotransposons autonomous for transposition to be identified in mammals. Their high rates of retrotransposition indicate that they are potent insertional mutagens that could serve as safe (noninfectious) genetic tools in a large panel of cells.

Animals↗

PubMed2004

Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians.

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.

Amino Acid Sequence↗