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Endogenous viral elements derived from non-retroviral RNA viruses were described in various animal genomes. Whether they have a biological function such as host immune protection against related viruses is a field of intense study. Here, we investigated the repertoire of endogenous flaviviral elements (EFVEs) inAedesmosquitoes, the vectors of arboviruses such as dengue and chikungunya viruses. Previous studies identified three EFVEs fromAe. albopictusand one fromAe. aegypticell lines. However, in-depth characterization of EFVEs in wild-type mosquito populations and individualsin vivohas not been performed. We detected the full-length DNA sequence of the previously described EFVEs and their respective transcripts in severalAe. albopictusandAe. aegyptipopulations from geographically distinct areas. However, EFVE-derived proteins were not detected by mass spectrometry. Using deep sequencing, we detected the production of piRNA-like small RNAs in antisense orientation, targeting the EFVEs and their flanking regionsin vivo. The EFVEs were integrated in repetitive regions of the mosquito genomes, and their flanking sequences varied among mosquito populations from different geographical regions. We bioinformatically predicted several new EFVEs from a VietnameseAe. albopictuspopulation and observed variation in the occurrence of those elements among mosquito populations. Phylogenetic analysis of anAe. aegyptiEFVE suggested that it integrated prior to the global expansion of the species and subsequently diverged among and within populations. Together, this study revealed substantial structural and nucleotide diversity of flaviviral integrations inAedesgenomes. Unraveling this diversity will help to elucidate the potential biological function of these EFVEs.ImportanceEndogenous viral elements (EVEs) are whole or partial viral sequences integrated in host genomes. Interestingly, some EVEs have important functions for host fitness and antiviral defense. Because mosquitoes also have EVEs in their genomes, we decided to thoroughly characterized them to lay the foundation of the potential use of these EVEs to manipulate the mosquito antiviral response. Here, we focused on EVEs related to theFlavivirusgenus, to which dengue and Zika viruses belong, inAedesmosquito individuals from geographically distinct areas. We showed the existencein vivoof flaviviral EVEs previously identified in mosquito cell lines and we detected new ones. We showed that EVEs have evolved differently in each mosquito population. They produced transcripts and small RNAs, but not proteins, suggesting a function at the RNA level. Our study uncovers the diverse repertoire of flaviviral EVEs inAedesmosquito populations and suggests a role in the host antiviral system.

Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51.

International audience; Human Immunodeficiency virus type 1 (HIV-1), as well as many other viruses that depend on nuclear entry for replication, has developed an evolutionary strategy to dock and translocate through the nuclear pore complex (NPC). In particular, the nuclear pore is not a static window but it is a dynamic structure involved in many vital cellular functions, as nuclear import/export, gene regulation, chromatin organization and genome stability. This review aims to shed light on viral mechanisms developed by HIV-1 to usurp cellular machinery to favor viral gene expression and their replication. In particular, it will be reviewed both what is known and what is speculated about the link between HIV translocation through the nuclear pore and the proviral integration in the host chromatin.

Upon antiretroviral therapy (ART) human immunodeficiency virus (HIV)/human T-cell lymphotropic virus type 1 (HTLV-1) co-infected individuals frequently develop neurological disorders through hitherto unknown mechanisms. Here, we show that effective anti-HIV ART increases HTLV-1 proviral load through a polyclonal integration pattern of HTLV-1 in both CD4+ and CD8+ T-cell subsets that is reminiscent of that typically associated with HTLV-1-related inflammatory conditions. These data indicate that preventing ART-triggered clonal expansion of HTLV-1-infected cells in co-infected individuals deserves investigation.

International audience; Integrated hepatitis B virus (HBV) DNA is found in the great majority of human hepatocellular carcinomas, suggesting that these viral integrations may be implicated in liver oncogenesis. Besides the insertional mutagenesis characterized in a few selected cases and the contribution of viral transactivators to cell transformation to malignancy, HBV has been shown to generate gross chromosomal rearrangements potentially involved in carcinogenesis. Here, we report a t(3;8) chromosomal translocation present in a hepatocellular carcinoma developed in noncirrhotic liver tissue. One side of the translocation, in 8p23, is shown to be in the vicinity of the carboxypeptidase N gene, a locus that is heavily transcribed in liver tissue and frequently deleted in hepatocellular carcinomas and other epithelial tumors. The other side of the translocation, in 3q27-29, is widely implicated in several types of translocations occurring in different malignancies, such as large-cell lymphomas. The present data strongly support a model in which HBV-induced chromosomal rearrangements play a key role during multistep liver oncogenesis.

BackgroundHuman pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods.ResultsWe used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration.ConclusionsWe have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also forin vitroapplications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

TNPO3 is a nuclear importer required for HIV-1 infection. Here, we show that depletion of TNPO3 leads to an HIV-1 block after nuclear import but prior to integration. To investigate the mechanistic requirement of TNPO3 in HIV-1 infection, we tested the binding of TNPO3 to the HIV-1 core and found that TNPO3 binds to the HIV-1 core. Overall, this work suggests that TNPO3 interacts with the incoming HIV-1 core in the cytoplasm to assist a process that is important for HIV-1 infection after nuclear import.

International audience; A novel extrachromosomal element that we called pAMT11 was discovered in a deep-sea vent isolate belonging to the hyperthermophilic euryarchaeal order Thermococcales. It consists of a double-stranded DNA of 20,534bp which encodes 30 putative open reading frames (ORFs) of which six could be assigned to a putative function on the basis of sequence similarity to known genes or to protein domain families. Most of the ORFs of pAMT1 showed homology and synteny with a genomic island of Thermococcus kodakaraensis KOD1. This region, named TKV1, was previously described as a "virus-like integrated element" and assumed to integrate into the host chromosome by a site-specific recombination mechanism similar to that of Sulfolobus solfataricus virus 1. While most of the genes shared by pAMT11 and TKV1 encode putative membrane proteins presumably involved in virus particle formation, attempts to induce production of virus particles by mitomycin treatment of AMT11 cultures failed, suggesting that pAMT11 may represent the genome of a defective virus or a plasmid. Genomes of mobile elements usually contain two regions: a core of conserved genes mainly involved in replication, maintenance or spreading of the genetic element, and a variable set of accessory genes. Surprisingly, genes presumably implied in the replication process are quite divergent between TKV1 and pAMT11. Indeed, TKV1 possesses a MCM-like protein that may function as a replication initiator, while pAMT11 encodes a putative non-conventional protein distantly related to the Rep protein previously described in a small plasmid of Pyrococcus sp. strain JT1, assumed to replicate by a rolling-circle (RC) mechanism. However, in the case of pAMT11, this mode of plasmid replication could not be experimentally proven and is questionable given the lack of significant similarities with any other members of the RC-Rep superfamily and its unusual large size compared to other RC plasmids.

International audience; Tailed double-stranded DNA viruses (order Caudovirales) represent the dominant morphotype among viruses infecting bacteria. Analysis and comparison of complete genome sequences of tailed bacterial viruses provided insights into their origin and evolution. Structural and genomic studies have unexpectedly revealed that tailed bacterial viruses are evolutionarily related to eukaryotic herpesviruses. Organisms from the third domain of life, Archaea, are also infected by viruses that, in their overall morphology, resemble tailed viruses of bacteria. However, high-resolution structural information is currently unavailable for any of these viruses, and only a few complete genomes have been sequenced so far. Here we identified nine proviruses that are clearly related to tailed bacterial viruses and integrated into chromosomes of species belonging to four different taxonomic orders of the Archaea. This more than doubled the number of genome sequences available for comparative studies. Our analyses indicate that highly mosaic tailed archaeal virus genomes evolve by homologous and illegitimate recombination with genomes of other viruses, by diversification, and by acquisition of cellular genes. Comparative genomics of these viruses and related proviruses revealed a set of conserved genes encoding putative proteins similar to virion assembly and maturation, as well as genome packaging proteins of tailed bacterial viruses and herpesviruses. Furthermore, fold prediction and structural modeling experiments suggest that the major capsid proteins of tailed archaeal viruses adopt the same topology as the corresponding proteins of tailed bacterial viruses and eukaryotic herpesviruses. Data presented in this study strongly support the hypothesis that tailed viruses infecting archaea share a common ancestry with tailed bacterial viruses and herpesviruses.

International audience; Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3' processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies.

International audience; Lentiviral vectors are under intense scrutiny as unique candidate viral vector vaccines against tumor and aggressive pathogens because of their ability to initiate potent and durable specific immune responses. Strategies that alleviate safety concerns will facilitate the clinical developments involving lentiviral vectors. In this respect, the development of integration deficient lentiviral vectors circumvents the safety concerns relative to insertional mutagenesis and might pave the way for clinical applications in which gene transfer is targeted to non-dividing cells. We thus evaluated the potential use of nonintegrative lentiviral vectors as vaccination tools since the main targeted cell in vaccination procedures is the non-dividing dendritic cell (DC). In this study, we demonstrated that a single administration of nonintegrative vectors encoding a secreted form of the envelope of a virulent strain of West Nile Virus (WNV) induces a robust B cell response. Remarkably, nonintegrative lentiviral vectors fully protected mice from a challenge with a lethal dose of WNV and a single immunization was sufficient to induce early and long-lasting protective immunity. Thus, nonintegrative lentiviral vectors might represent a safe and efficacious vaccination platform for the development of prophylactic vaccines against infectious agents.

Free in PubMed Central : http://www.pubmedcentral.nih.gov/picrender.fcgi?tool=pmcentrez&artid=1951289&blobtype=pdf; The heterodimer Ku70/80 Ku is the DNA-binding component of the DNA-PK complex required for the nonhomologous end-joining pathway. It participates in numerous nuclear processes, including telomere and chromatin structure maintenance, replication, and transcription. Ku interacts with retroviral preintegration complexes and is thought to interfere with the retroviral replication cycle, in particular the formation of 2-long terminal repeat (LTR) viral DNA circles, viral DNA integration, and transcription. We describe here the effect of Ku80 on both provirus integration and the resulting transgene expression in cells transduced with retroviral vectors. We found that transgene expression was systematically higher in Ku80-deficient xrs6 cells than in Ku80-expressing CHO cells. This higher expression was observed irrespective of the presence of the viral LTR and was also not related to the nature of the promoter. Real-time PCR monitoring of the early viral replicative steps demonstrated that the absence of Ku80 does not affect the efficiency of transduction. We analyzed the transgene distributions localization in nucleus by applying a three-dimensional reconstruction model to two-dimensional fluorescence in situ hybridization images. This indicated that the presence of Ku80 resulted in a bias toward the transgenes being located at the periphery of the nucleus associated with their being repressed; in the absence of this factor the transgenes tend to be randomly distributed and actively expressed. Therefore, although not strictly required for retroviral integration, Ku may be involved in targeting retroviral elements to chromatin domains prone to gene silencing.

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.

Retroviral integration is central to viral persistence and pathogenesis, cancer as well as host genome evolution. However, it is unclear why integration appears essential for retrovirus production, especially given the abundance and transcriptional potential of non-integrated viral genomes. The involvement of retroviral endonuclease, also called integrase (IN), in replication steps apart from integration has been proposed, but is usually considered to be accessory. We observe here that integration of a retrovirus from the spumavirus family depends mainly on the quantity of viral DNA produced. Moreover, we found that IN directly participates to linear DNA production from 2-LTR circles by specifically cleaving the conserved palindromic sequence found at LTR-LTR junctions. These results challenge the prevailing view that integrase essential function is to catalyze retroviral DNA integration. Integrase activity upstream of this step, by controlling linear DNA production, is sufficient to explain the absolute requirement for this enzyme. The novel role of IN over 2-LTR circle junctions accounts for the pleiotropic effects observed in cells infected with IN mutants. It may explain why 1) 2-LTR circles accumulate in vivo in mutants carrying a defective IN while their linear and integrated DNA pools decrease; 2) why both LTRs are processed in a concerted manner. It also resolves the original puzzle concerning the integration of spumaretroviruses. More generally, it suggests to reassess 2-LTR circles as functional intermediates in the retrovirus cycle and to reconsider the idea that formation of the integrated provirus is an essential step of retrovirus production.

Integration of the human papillomavirus (HPV) genome into the host genome is associated with the disruption of the HPV E2 gene and with amplification and rearrangement of the viral and flanking cellular sequences. Molecular characterization of the genomic structures of coamplified HPV sequences and oncogenes provides essential information concerning the mechanisms of amplification and their roles in carcinogenesis. Using fluorescent hybridization on stretched DNA molecules in two cervical cancer–derived cell lines, we have elucidated the genomic structures of amplified regions containing HPV/myc genes over several hundreds of kilobases. Direct visualization of hybridization signals on individual DNA molecules suggests that overreplication and breakage-fusion-bridge–type mechanisms are involved in the genomic instability associated with HPV cervical cancers. Further analysis from two other genital cancer–derived cell lines reveals a recurrent motif of amplification, probably generated by a common mechanism involving overreplication upon viral integration. Interestingly, different amplification patterns seem to be correlated with the disease outcome, thus providing new insights into HPV-related cancer development and tumor progression.

International audience; Retroviral infection triggers the cytoplasmic translocation of two Crm1-dependent shuttle factors, namely the Ini1 (integrase interactor 1, hSNF5) and the promyelocytic leukemia (PML) protein. Blocking nuclear export of shuttle factors by leptomycin B increases the efficiency of retroviral integration, suggesting that some may mediate antiviral activity. While PML was shown to counteract proviral establishment, it remained unclear whether Ini1, a protein implicated in various processes during human immunodeficiency virus replication, has the same potential. Employing RNA interference-mediated knock-down of Ini1, we show here that the simultaneous accumulation of both proteins in the cytoplasm likely reflects two non-interdependent phenomena. Furthermore, Ini1 does not interfere with retroviral integration, as cells lacking Ini1 show no increased infection susceptibility.

Viral integrase (IN) and Vpr are both components of the human immunodeficiency virus type 1 (HIV-1) pre-integration complex. To investigate whether these proteins interact within this complex, we investigated the effects of Vpr and its subdomains on IN activity in vitro. When a 21mer oligonucleotide was used as a donor and acceptor, both Vpr and its C-terminal DNA-binding domain [(52-96)Vpr] inhibited the integration reaction, whereas the (1-51)Vpr domain did not affect IN activity. Steady-state fluorescence anisotropy showed that both full-length and (52-96)Vpr bind to the short oligonucleotide, thereby extending previous observations with long DNA. The concentrations of the two proteins required to inhibit IN activity were consistent with their affinities for the oligonucleotide. The use of a 492 bp mini-viral substrate confirmed that Vpr can inhibit the IN-mediated reaction. However, the activity of (52-96)Vpr differed notably since it stimulated specifically integration events involving two homologous mini-viral DNAs. Order of addition experiments indicated that the stimulation was maximal when IN, (50-96)Vpr and the mini-viral DNA were allowed to form a complex. Furthermore, in the presence of (50-96)Vpr, the binding of IN to the mini-viral DNA was dramatically enhanced. Taken together, these data suggest that (52-96)Vpr stimulates the formation of a specific complex between IN and the mini-viral DNA.

International audience; Integration of HIV-1 genome into host cell chromosome is mediated by viral integrase (IN). The IN catalytic core (CC, IN(50-212)) dimerizes through mutual interactions of its alpha1 and alpha5 helices. Peptides INH1 and INH5 reproducing these helix sequences strongly inhibited IN. For instance, an IC(50) of 80 nM was determined for INH5 in integration assays using wild-type IN (wtIN). In size exclusion chromatography, INH1 and INH5 perturbed the association-dissociation equilibrium of both dmIN (IN(1-288)/F185K/C280S) and CC, leading to monomers as surviving species, while in circular dichroism, binding of peptides to dmIN altered the protein conformation. Thus, enzyme deactivation, subunit dissociation, and protein unfolding are events which parallel one another. The target of INH5 in the enzyme was then identified. In fluorescence spectroscopy, C(0.5) values of 168 and 44 nM were determined for the binding affinity of INH5 to IN and CC, respectively, at 115 nM subunit concentration, while interaction of INH5 with INH1 was found stronger than interaction of INH5 with itself (23 times larger in term of dissociation constants). These results strongly suggested that the alpha1 helix is the privileged target of INH5. The latter could serve as a lead for the development of new chemotherapeutic agents against HIV-1.

The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).

International audience; The human immunodeficiency virus type-1 (HIV-1) integrase is known to form a highly stable interaction with the termini of the linear, pre-integrated retroviral genome, where it catalyzes the 3'-OH processing and strand transfer processes required for their coordinated integration into host DNA. Here, we determine that the association of HIV-1 integrase with the viral DNA termini leads to the formation of two classes of nucleoprotein complexes with distinct properties in vitro. Both bound states are intrinsically stable and highly resistant to exonuclease digestion, but nonetheless they exhibit different stabilities in the presence of single-stranded polynucleotides. While a population of preassembled complexes tolerates elevated polynucleotide concentrations, the remainder forms an unstable ternary (integrase-substrate-polynucleotide) intermediate, leading to the rapid expulsion of the otherwise tightly bound substrate. The distribution of complexes between the two states is influenced by the preincubation time and temperature, increases in either of which favor the formation of the challenge-resistant species. Challenge-resistant complexes are formed more efficiently with Mn2+ than with Mg2+ and are sensitive to the length rather than the sequence of the DNA substrate. Due to the delayed appearance of the challenge-resistant form after the initial stable binding of the DNA substrate, our results may be indicative of a structural change in the preassembled complex which thereby modulates its response to exogenous DNA targets.

International audience; Hepatitis B virus (HBV) DNA is found chromosomally integrated into the genome of the majority of hepatocellular carcinomas (HCC) arising in chronic HBV carriers suggesting that, in some instances, viral sequences may be directly responsible for oncogenic conversion. In an attempt to clarify the oncogenic potential of integrated HBV sequences, we performed an extensive analysis of two single integrations present in HCC which developed in non-cirrhotic livers from HBsAg-positive Korean patients. In both cases, integrated viral sequences were characterized by a duplicated-inverted configuration involving the flanking cellular sequences, a pattern consistently found in many amplicons isolated from mammalian cells. Integration sites are characterized by an AT-rich content and the presence of topoisomerase I and II cleavage target sequences as well as other recombination-prone motifs. The chromosomal locations of the integration sites were determined as 8q13 and 10q22 in the human genome, two regions known to harbour genes involved in tumorigenesis. The cis-activating potential of the integrations in their original configuration was also investigated in a transient transfection assay in HepG2 cells. Integrated sequences, rather than activating heterologous promoters, show either no activity or a weak tendency to inhibit activation of neighbouring reporter genes. The implications of our findings for the understanding of primary liver cancer development are discussed.