[Show abstract][Hide abstract] ABSTRACT: Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation.
Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication.
Our data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.
[Show abstract][Hide abstract] ABSTRACT: LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction.
We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket.
Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post- integration production of infectious viral particles takes place.
[Show abstract][Hide abstract] ABSTRACT: Nef is a Human immunodeficiency virus type-1 (HIV-1) auxiliary protein, which plays an important role in virus replication and the onset of acquired immunodeficiency. Although known functions of Nef might explain its contribution to HIV-1 associated pathogenesis, how Nef increases virus infectivity is still an open question. In vitro, Nef-deleted viruses have a defect that prevents efficient completion of early steps of replication. We have previously shown that this restriction is not due to the absence of Nef in viral particles. Rather, a loss of function in virus-producing cells accounts for the lower infectivity of nef-deleted viruses as compared with wild-type (WT) viruses. Here we used DiGE and iTRAQ to identify differences between the proteomes of WT and nef-deleted viruses. We observe that glucosidase II is enriched in WT virions whereas Ezrin, ALG-2, CD81 and EHD4 are enriched in nef-deleted virions. Functional analysis shows that glucosidase II, ALG-2 and CD81 have no or only Nef-independent effect on infectivity. By contrast, Ezrin and EHD4 are involved in the ability of Nef to increase virus infectivity (referred to thereafter as Nef potency). Indeed, simultaneous Ezrin and EHD4 depletion in SupT1 and 293T virus-producing cells result in a ∼30 and ∼70% decrease of Nef potency, respectively. Finally, while Ezrin behaves as an inhibitory factor counteracted by Nef, EHD4 should be considered as a co-factors required by Nef to increase virus infectivity.
Journal of Virology 01/2013; 87(7). DOI:10.1128/JVI.02477-12 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Simian foamy viruses (SFV) are widespread retroviruses among non-human primates (NHP). SFV actively replicates in their oral cavity and can be transmitted to humans after NHP bites, giving rise to a persistent infection even decades after primary infection. Very few data are available on the genetic structure of such SFV found in humans.In the framework of ongoing studies searching for SFV-infected humans in South Cameroon rainforest villages, we studied 38 SFV-infected hunters whose time of infection were presumably determined. By long-term co-cultures of PBMCs with BHK-21 cells, we isolated 5 new SFV strains, providing complete genomes of SFVcpz Pan troglodytes troglodytes (BAD327, AG15), SFVcni Cercopithecus nictitans (AG16) and SFVggo Gorilla gorilla (BAK74 and BAD468). These zoonotic strains share a very high similarity with their NHP counterparts with a high conservation of the genetic elements important for viral replication. Interestingly, FV DNA sequences obtained before cultivation revealed in both U3 and tas some deletion variants that may correlate with in vivo chronicity in humans. Genomic changes in bet (premature stop codon) and gag were also observed. To know if such changes were specific to zoonotic strains, we studied local SFV-infected chimpanzees and found the same genomic changes.Our study reveals that natural polymorphism of SFV strains does exist, at both inter-subspecies level (gag, bet) and intra-subspecies level (U3, tas) but seems not to reflect a viral adaptation specific to zoonotic SFV strains.
Journal of Virology 09/2012; 86(24). DOI:10.1128/JVI.01715-12 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: L’intégration du génome viral dans le génome de la cellule hôte est une étape indispensable à la réplication des rétroéléments, vecteurs adaptés à la thérapie génique mais aussi agents mutagènes potentiels. Récemment, de nombreuses études ont montré que les sites d’intégration ne sont pas distribués aléatoirement sur le génome mais, au contraire, préférentiellement localisés dans certaines régions, démontrant que la spécificité d’intégration est un mécanisme hautement régulé. Plusieurs protéines virales et facteurs cellulaires jouent un rôle fondamental dans l’étape d’intégration, certains d’entre eux participant également à la spécificité d’intégration. Cette revue décrit les avancées récentes sur l’intégration des rétroéléments, se focalisant sur les mécanismes impliqués dans la sélectivité et la spécificité d’intégration, ainsi que dans l’étape d’ancrage chromatinien précédant l’insertion du provirus.
[Show abstract][Hide abstract] ABSTRACT: Small regions called protein transduction domains (PTDs), identified in cellular and viral proteins, have been reported to efficiently cross biological membranes. Here we show that the structural Gag protein of the prototypic foamy virus (PFV) is apparently able to move from cell to cell and to transport the green fluorescent protein (GFP) from few transfected cells to the nuclei of the entire monolayer. Deletion studies showed that this property lies within the second glycine/arginine (GRII) box in the C-terminus of the protein. We also found that uptake and nuclear accumulation of Gag GRII expressed as GFP-fusion protein in recipient cells was observed only following methanol fixation, but never in living cells or when cells were fixed with glutaraldehyde or treated with trichloroacetic acid prior to methanol fixation. Absence of intercellular spreading in vivo was further confirmed using a sensitive luciferase activity assay based on transactivation of the PFV long terminal repeats. Thus, we conclude that intercellular spreading of PFV Gag represents an artificial diffusion event occurring during cell fixation, followed by nuclear retention mediated by the chromatin-binding sequence within the Gag GRII box. In light of these results, we advise caution before defining a peptide as PTD on the basis of intercellular spreading observed by fluorescence microscopy.
PLoS ONE 02/2012; 7(2):e31108. DOI:10.1371/journal.pone.0031108 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to characterize simian foamy retroviruses (SFVs) in wild-born nonhuman primates (NHPs) in Gabon and to investigate
cross-species transmission to humans, we obtained 497 NHP samples, composed of 286 blood and 211 tissue (bush meat) samples.
Anti-SFV antibodies were found in 31 of 286 plasma samples (10.5%). The integrase gene sequence was found in 38/497 samples,
including both blood and tissue samples, with novel SFVs in several Cercopithecus species. Of the 78 humans, mostly hunters, who had been bitten or scratched by NHPs, 19 were SFV seropositive, with 15 cases
confirmed by PCR. All but one were infected with ape SFV. We thus found novel SFV strains in NHPs in Gabon and high cross-species
transmission of SFVs from gorilla bites.
Journal of Virology 11/2011; 86(2):1255-60. DOI:10.1128/JVI.06016-11 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HIV-1 integrase (IN) orchestrates the integration of the reverse transcribed viral cDNA into the host cell genome and participates also in other steps of HIV-1 replication. Cellular and viral factors assist IN in performing its multiple functions, and post-translational modifications contribute to modulate its activities. Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO acceptor sites. Viruses harboring SUMOylation site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Because SUMOylation-defective IN mutants retained WT catalytic activity, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication.
[Show abstract][Hide abstract] ABSTRACT: The Gag polyproteins play distinct roles during the replication cycle of retroviruses, hijacking many cellular machineries to fulfill them. In the case of the prototype foamy virus (PFV), Gag structural proteins undergo transient nuclear trafficking after their synthesis, returning back to the cytoplasm for capsid assembly and virus egress. The functional role of this nuclear stage as well as the molecular mechanism(s) responsible for Gag nuclear export are not understood.
We have identified a leptomycin B (LMB)-sensitive nuclear export sequence (NES) within the N-terminus of PFV Gag that is absolutely required for the completion of late stages of virus replication. Point mutations of conserved residues within this motif lead to nuclear redistribution of Gag, preventing subsequent virus egress. We have shown that a NES-defective PFV Gag acts as a dominant negative mutant by sequestrating its wild-type counterpart in the nucleus. Trans-complementation experiments with the heterologous NES of HIV-1 Rev allow the cytoplasmic redistribution of FV Gag, but fail to restore infectivity.
PFV Gag-Gag interactions are finely tuned in the cytoplasm to regulate their functions, capsid assembly, and virus release. In the nucleus, we have shown Gag-Gag interactions which could be involved in the nuclear export of Gag and viral RNA. We propose that nuclear export of unspliced and partially spliced PFV RNAs relies on two complementary mechanisms, which take place successively during the replication cycle.
[Show abstract][Hide abstract] ABSTRACT: Each of the pathogenic human retroviruses (HIV-1/2 and HTLV-1) has a nonhuman primate counterpart, and the presence of these retroviruses in humans results from interspecies transmission. The passage of another simian retrovirus, simian foamy virus (SFV), from apes or monkeys to humans has been reported. Mandrillus sphinx, a monkey species living in central Africa, is naturally infected with SFV. We evaluated the natural history of the virus in a free-ranging colony of mandrills and investigated possible transmission of mandrill SFV to humans.
We studied 84 semi-free-ranging captive mandrills at the Primate Centre of the Centre International de Recherches Médicales de Franceville (Gabon) and 15 wild mandrills caught in various areas of the country. The presence of SFV was also evaluated in 20 people who worked closely with mandrills and other nonhuman primates. SFV infection was determined by specific serological (Western blot) and molecular (nested PCR of the integrase region in the polymerase gene) assays. Seropositivity for SFV was found in 70/84 (83%) captive and 9/15 (60%) wild-caught mandrills and in 2/20 (10%) humans. The 425-bp SFV integrase fragment was detected in peripheral blood DNA from 53 captive and 8 wild-caught mandrills and in two personnel. Sequence and phylogenetic studies demonstrated the presence of two distinct strains of mandrill SFV, one clade including SFVs from mandrills living in the northern part of Gabon and the second consisting of SFV from animals living in the south. One man who had been bitten 10 years earlier by a mandrill and another bitten 22 years earlier by a macaque were found to be SFV infected, both at the Primate Centre. The second man had a sequence close to SFVmac sequences. Comparative sequence analysis of the virus from the first man and from the mandrill showed nearly identical sequences, indicating genetic stability of SFV over time.
Our results show a high prevalence of SFV infection in a semi-free-ranging colony of mandrills, with the presence of two different strains. We also showed transmission of SFV from a mandrill and a macaque to humans.
[Show abstract][Hide abstract] ABSTRACT: The human T-lymphotropic virus type I oncoprotein Tax is critical for T-cell transformation, acting mainly through nuclear factor kappa B essential modulator (NEMO) binding and subsequent nuclear factor-κB activation. Tax localizes to Tax nuclear bodies and to the centrosome and is subjected to ubiquitylation and small ubiquitin-like modifier (SUMO)ylation, which are both necessary for complete transcriptional activation. Using the photoconvertible fluorophore Dendra-2 coupled with live video confocal microscopy, we show for the first time that the same Tax molecule shuttles among Tax nuclear bodies and between these nuclear bodies and the centrosome, depending on its posttranslational modifications. Ubiquitylation targets Tax to nuclear bodies to which NEMO is recruited and subsequently SUMOylated. We also demonstrate that Tax nuclear bodies contain the SUMOylation machinery including SUMO and the SUMO conjugating enzyme Ubc9, strongly suggesting that these nuclear bodies represent sites of active SUMOylation. Finally, both ubiquitylation and SUMOylation of Tax control NEMO targeting to the centrosome. Altogether, we are proposing a model where both ubiquitylation and SUMOylation of Tax control the shuttling of Tax and NEMO between the cytoplasmic and nuclear compartments.
[Show abstract][Hide abstract] ABSTRACT: Les modifications post-traductionnelles, telles que la SUMOylation, sont un exemple de machinerie cellulaire détournée par les virus. La SUMOylation, qui consiste en la liaison covalente d'un peptide SUMO (small ubiquitin-like modifier) à une protéine cible, a récemment été impliquée dans le cycle réplicatif de nombreux virus. Cette revue rappelle, par des exemples choisis à travers le monde viral et en insistant sur les dernières découvertes, les différentes façons dont la SUMOylation est détournée par les virus afin de créer un environnement favorable à la réplication. Le lien entre les pathologies impliquées par chaque virus et la SUMOylation est abordé. Enfin, les dernières avancées concernant l'utilisation des potentiels inhibiteurs de la SUMOylation dans le cadre de la lutte antivirale et anticancéreuse sont évoquées.
[Show abstract][Hide abstract] ABSTRACT: Although viral RNA constitutes the majority of nucleic acids packaged in virions, a late occurring step of reverse transcription leads to the presence of infectious viral cDNA in foamy virus particles. This peculiarity distinguishes them from the rest of the retroviral family.
To evaluate the respective contribution of these viral nucleic acids in the replication of foamy viruses, their fate was studied by real-time PCR and RT-PCR early after infection, in the presence or in the absence of AZT. We found that an early reverse transcription step, which occurs during the first hours post-entry, is absolutely required for productive infection. Remarkably, sensitivity to AZT can be counteracted by increasing the multiplicity of infection (moi). We also show that 2-LTR circular viral DNA, which appears as soon as four hours post-infection, is transcriptionally competent.
Taken together, our data demonstrate that an early reverse transcription process, which takes place soon after viral entry, is indispensable for infectivity of FVs at low moi, when the amount of DNA-containing particles is not sufficient to lead to a productive infection. This study demonstrates a key role of the packaged viral RNA in the foamy virus infection, suggesting that the replication of this virus can be achieved by involving either viral DNA or RNA genome, depending on the condition of infection.
PLoS ONE 06/2010; 5(6):e11023. DOI:10.1371/journal.pone.0011023 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since integration into the host cell genome is an obligatory step for their replication, retro-elements are both potent insertional mutagens and also suitable vectors for gene therapy. Many recent studies reported that the integration process is not random but, on the contrary, higly regulated at the molecular level. Many viral proteins and cellular factors play a key role in the integration step, explaining the reason why different retro-elements display distinct integration profiles. This review describes the recent highlights about integration of retro-elements with particular focus on the mechanisms underlying the specificity of integration target-site selection and the step of chromosomal tethering which preceeds insertion of the provirus.
[Show abstract][Hide abstract] ABSTRACT: The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.