VSV-G pseudotyping rescues HIV-1 CA mutations that impair core assembly or stability

<p>Abstract</p> <p>Background</p> <p>The machinery of early HIV-1 replication still remains to be elucidated. Recently the viral core was reported to persist in the infected cell cytoplasm as an assembled particle, giving rise to the reverse transcription complex responsible for the synthesis of proviral DNA and its transport to the nucleus. Numerous studies have demonstrated that reverse transcription of the HIV-1 genome into proviral DNA is tightly dependent upon proper assembly of the capsid (CA) protein into mature cores that display appropriate stability. The functional impact of structural properties of the core in early replicative steps has yet to be determined.</p> <p>Results</p> <p>Here, we show that infectivity of HIV-1 mutants bearing S₁₄₉A and S₁₇₈A mutations in CA can be efficiently restored when pseudotyped with vesicular stomatitis virus envelope glycoprotein, that addresses the mutant cores through the endocytic pathway rather than by fusion at the plasma membrane. The mechanisms by which these mutations disrupt virus infectivity were investigated. S₁₄₉A and S₁₇₈A mutants were unable to complete reverse transcription and/or produce 2-LTR DNA. Morphological analysis of viral particles and <it>in vitro </it>uncoating assays of isolated cores demonstrated that infectivity defects resulted from disruption of the viral core assembly and stability for S₁₄₉A and S₁₇₈A mutants, respectively. Consistent with these results, both mutants failed to saturate TRIM-antiviral restriction activity.</p> <p>Conclusion</p> <p>Defects generated at the level of core assembly and stability by S₁₄₉A and S₁₇₈A mutations are sensitive to the way of delivery of viral nucleoprotein complexes into the target cell. Addressing CA mutants through the endocytic pathway may compensate for defects generated at the reverse transcription/nuclear import level subsequent to impairment of core assembly or stability.</p

Predominant role of Tax sumoylation in Tax-induced NF-kB activation in T cells

International audiencen.

The Scaffolding Protein Dlg1 Is a Negative Regulator of Cell-Free Virus Infectivity but Not of Cell-to-Cell HIV-1 Transmission in T Cells

Background: Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before. Methodology/Principal Findings: Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA

Etude du rôle de la protéine Dlg1 dans le cycle de réplication et la transmission du VIH-1

La propagation du VIH-1 dans l’organisme est un processus dépendant du bourgeonnement de particules correctement assemblées. Pour ce faire, le virus utilise une stratégie de détournement de certaines protéines cellulaires, qui sont souvent des partenaires des protéines de structure virale Gag et Env. Le VIH-1 se transmet de deux façons dans un organisme : par particules libres ou par transfert de cellule à cellule via une structure appelée synapse virologique. Ce dernier moyen de propagation est considéré comme le plus efficace et prédominant in vitro. Nous avons précédemment identifié Dlg1, l’homologue humain de la protéine « Discs Large » de la drosophile, comme un nouveau partenaire de la protéine Gag du VIH-1. Dans ce contexte, nous avons étudié le rôle de Dlg1 dans les deux modes de transmission du VIH-1 dans des lymphocytes T, cellules cibles naturelles du virus, où l’extinction stable de Dlg1 est obtenue avec un vecteur shRNA. Nous avons montré que le virus se propage plus efficacement dans les cellules n’exprimant pas Dlg1. Des observations en microscopie confocale nous ont révélé que l’absence de Dlg1 n’empêchait pas la formation de la synapse virologique, et des analyses quantitatives nous ont montré que l’efficacité de la transmission de cellule-à-cellule du virus n’était également pas affectée. La meilleure efficacité de propagation en absence de Dlg1 résulte d’une augmentation significative de l’infectivité des particules relâchées. Ce phénotype des particules virales correspond à une augmentation de leur contenu en cholestérol qui permettrait d’augmenter l’efficacité de la fusion virus-cellule lors de l’étape d’entrée du virus. Nous avons donc montré que Dlg1 est un régulateur négatif de la transmission par particules libres du VIH-1, et que la transmission cellule-à-cellule du virus parvient à contourner cette régulation

Etude du rôle de la protéine Dlg1 dans le cycle de réplication et la transmission du VIH-1

La propagation du VIH-1 dans l’organisme est un processus dépendant du bourgeonnement de particules correctement assemblées. Pour ce faire, le virus utilise une stratégie de détournement de certaines protéines cellulaires, qui sont souvent des partenaires des protéines de structure virale Gag et Env. Le VIH-1 se transmet de deux façons dans un organisme : par particules libres ou par transfert de cellule à cellule via une structure appelée synapse virologique. Ce dernier moyen de propagation est considéré comme le plus efficace et prédominant in vitro. Nous avons précédemment identifié Dlg1, l’homologue humain de la protéine « Discs Large » de la drosophile, comme un nouveau partenaire de la protéine Gag du VIH-1. Dans ce contexte, nous avons étudié le rôle de Dlg1 dans les deux modes de transmission du VIH-1 dans des lymphocytes T, cellules cibles naturelles du virus, où l’extinction stable de Dlg1 est obtenue avec un vecteur shRNA. Nous avons montré que le virus se propage plus efficacement dans les cellules n’exprimant pas Dlg1. Des observations en microscopie confocale nous ont révélé que l’absence de Dlg1 n’empêchait pas la formation de la synapse virologique, et des analyses quantitatives nous ont montré que l’efficacité de la transmission de cellule-à-cellule du virus n’était également pas affectée. La meilleure efficacité de propagation en absence de Dlg1 résulte d’une augmentation significative de l’infectivité des particules relâchées. Ce phénotype des particules virales correspond à une augmentation de leur contenu en cholestérol qui permettrait d’augmenter l’efficacité de la fusion virus-cellule lors de l’étape d’entrée du virus. Nous avons donc montré que Dlg1 est un régulateur négatif de la transmission par particules libres du VIH-1, et que la transmission cellule-à-cellule du virus parvient à contourner cette régulation

Loss of infectivity of HIV-1 particles produced by mobile lymphocytes.

HIV-1 spreads by cell-free particles and through direct cell contacts. To discriminate between these two modes of dissemination, an assay in which the cells are cultured under shaking conditions impairing cell-to-cell transmission has been described. We addressed the impact of shaking on HIV-1 particle infectivity. Kinetics of HIV-1 infection in static or shaking conditions confirmed that HIV-1 replication is reduced in mobile lymphocyte T cells. Strikingly, the infectivity of viruses produced by mobile lymphocytes was dramatically reduced. In parallel, the amount of envelope protein present on these particles showed a continuous decrease over time. We conclude that inefficient HIV-1 replication in mobile lymphocytes in this experimental system is not only due to avoidance of viral cell-to-cell transfer but also to the loss of infectivity of the viral particles due to the alteration of the composition and functionality of the particles produced by these lymphocytes. It is important to take these observations into account when studying viral transmission under shaking conditions

pH-dependent entry of chikungunya virus into Aedes albopictus cells

International audienceBackground: The chikungunya virus (CHIKV) recently caused explosive outbreaks in Indian Ocean islands and India. During these episodes, the virus was mainly spread to humans through the bite of the mosquito Aedes albopictus. Concomitantly to the description of symptoms of an unexpected severity in infants and elderly patients, a viral genome microevolution has been highlighted, in particular consisting in the acquisition of an A226V mutation in the gene encoding envelope glycoprotein E1, which was later found to confer an increased fitness for A. albopictus. We previously decrypted the entry pathway used by CHIKV to infect human epithelial cells and showed that these mechanisms are modulated by the E1-A226V mutation. In this report we investigated the conditions for CHIKV entry into mosquito cells and we assessed the consequence of E1 gene mutation on these parameters.Principal findings: Our main findings indicate that CHIKV infection of A. albopictus cell lines is sensitive to Bafilomycin A1 and chloroquine and to membrane cholesterol depletion. The E1-226V mutated LR-OPY1 isolate collected during the 2005 outbreak in La Réunion replicated more efficiently than the 37997 African reference strain in C6/36 cells. Moreover, the LR-OPY1 strain displayed greater membrane cholesterol dependence and was more sensitive to inhibition of endosomal pH acidification. Finally, using electron microscopy, we imaged CHIKV entry into C6/36 cells.Conclusions: Our data support that CHIKV is endocyted into A. albopictus cells and requires membrane cholesterol as well as a low-pH environment for entry. These features are modulated in some extent by the A226V mutation in the E1 gene of the LR-OPY1 isolate. Altogether, our data provide information regarding the pathways used by CHIKV to infect A. albopictus cells

Viral entry route determines how human plasmacytoid dendritic cells produce type I interferons

International audienceAlthough plasmacytoid dendritic cells (pDCs) represent a rare immune cell type, they are the most important source of type I interferons (IFNs) upon viral infection. Phagocytosed RNA viruses and RNA virus–infected cells are detected by pDCs with the endosomal pattern recognition receptor (PRR) toll-like receptor 7 (TLR7). We showed that replication of the yellow fever live vaccine YF-17D in human pDCs and pDC-like cell lines stimulated type I IFN production through RIG-I (retinoic acid–inducible gene I), a member of the RIG-I–like receptor (RLR) family of cytosolic PRRs. Thus, human pDCs sense replicative viral RNA. In contrast, direct contact between pDCs and YF-17D–infected cells stimulated a TLR7-dependent, viral replication–independent production of type I IFN. We also showed that the RLR pathway was dampened by the activities of interleukin-1 receptor–associated kinases 1 and 4 (IRAK1 and IRAK4), which are downstream effectors of the TLR7 pathway, suggesting that both kinases play opposing roles downstream of specific PRRs. Together, these data suggest that a virus can stimulate either TLR or RLR signaling in the same cell, depending on how its nucleic acid content is delivered

Genomic diversity contributes to the neuroinvasiveness of the Yellow fever French neurotropic vaccine

International audienceMass vaccination with the live attenuated vaccine YF-17D is the current way to prevent infection with Yellow fever virus (YFV). However, 0.000012-0.00002% of vaccinated patients develop post-vaccination neurological syndrome (YEL-AND). Understanding the factors responsible for neuroinvasion, neurotropism, and neurovirulence of the vaccine is critical for improving its biosafety. The YF-FNV vaccine strain, known to be associated with a higher frequency of YEL-AND (0.3-0.4%) than YF-17D, is an excellent model to study vaccine neuroinvasiveness. We determined that neuroinvasiveness of YF-FNV occured both via infection and passage through human brain endothelial cells. Plaque purification and next generation sequencing (NGS) identified several neuroinvasive variants. Their neuroinvasiveness was not higher than that of YF-FNV. However, rebuilding the YF-FNV population diversity from a set of isolated YF-FNV-N variants restored the original neuroinvasive phenotype of YF-FNV. Therefore, we conclude that viral population diversity is a critical factor for YFV vaccine neuroinvasiveness