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

Medjez II

Ce gisement princeps du faciès « Sétifïen* » joue un rôle majeur dans la caractérisation de la variabilité capsienne de l’Epipaléolithique du Maghreb, observée entre le VIII-Ve millénaire cal BC. On doit à Henriette Camps-Fabrer d’avoir souligné les éléments diagnostics d’une abondante et nouvelle documentation régionale. Recueillie lors de plusieurs campagnes de fouilles (entre 1963 et 1968), cette documentation est conservée à Alger, au CNRPAH. Bien après la première monographie sur le Caps..

Uracil DNA Glycosylase 2 negatively regulates HIV-1 LTR transcription

Numerous cellular factors belonging to the DNA repair machineries, including RAD18, RAD52, XPB and XPD, have been described to counteract human immunodeficiency virus type 1 (HIV-1) replication. Recently, Uracil DNA glycosylase 2 (UNG2), a major determinant of the uracil base excision repair pathway, was shown to undergo rapid proteasome-dependent degradation following HIV-1 infection. However, the specific role of intracellular UNG2 depletion during the course of HIV-1 infection is not clearly understood. Our study shows for the first time that overexpression of UNG2 inhibits HIV-1 replication. We demonstrate that this viral inhibition is correlated with a marked decrease in transcription efficiency as shown by monitoring HIV-1 LTR promoter activity and quantification of HIV-1 RNA levels. Interestingly, UNG2 inhibits LTR activity when stimulated by Tat transactivator or TNFα, while barely affected using Phorbol ester activation. Mutational analysis of UNG2 indicates that antiviral activity may require the integrity of the UNG2 catalytic domain. Altogether, our data indicate that UNG2 is likely to represent a new host defense factor specifically counteracted by HIV-1 Vpr. The molecular mechanisms involved in the UNG2 antiviral activity still remain elusive but may rely on the sequestration of specific cellular factor(s) critical for viral transcription

Patterns of HIV-1 Protein Interaction Identify Perturbed Host-Cellular Subsystems

Human immunodeficiency virus type 1 (HIV-1) exploits a diverse array of host cell functions in order to replicate. This is mediated through a network of virus-host interactions. A variety of recent studies have catalogued this information. In particular the HIV-1, Human Protein Interaction Database (HHPID) has provided a unique depth of protein interaction detail. However, as a map of HIV-1 infection, the HHPID is problematic, as it contains curation error and redundancy; in addition, it is based on a heterogeneous set of experimental methods. Based on identifying shared patterns of HIV-host interaction, we have developed a novel methodology to delimit the core set of host-cellular functions and their associated perturbation from the HHPID. Initially, using biclustering, we identify 279 significant sets of host proteins that undergo the same types of interaction. The functional cohesiveness of these protein sets was validated using a human protein-protein interaction network, gene ontology annotation and sequence similarity. Next, using a distance measure, we group host protein sets and identify 37 distinct higher-level subsystems. We further demonstrate the biological significance of these subsystems by cross-referencing with global siRNA screens that have been used to detect host factors necessary for HIV-1 replication, and investigate the seemingly small intersect between these data sets. Our results highlight significant host-cell subsystems that are perturbed during the course of HIV-1 infection. Moreover, we characterise the patterns of interaction that contribute to these perturbations. Thus, our work disentangles the complex set of HIV-1-host protein interactions in the HHPID, reconciles these with siRNA screens and provides an accessible and interpretable map of infection

Etude de l'étape d'entrée des vecteurs lentiviraux dérivés du VIH-1 dans les cellules hématopoïétiques humaines

Les vecteurs lentiviraux (LV) sont des outils efficaces de transfert de gène, largement utilisés en thérapie génique, en particulier pour la transduction ex vivo de cellules souches et progénitrices hématopoïétiques (CSPH). Afin d étudier simultanément la fusion et la transduction dans les CSPH avec les LV, nous avons adapté une méthode basée sur latechnologie du transfert d énergie entre deux molécules fluorescentes (FRET). Pour mettre en place cette technique, des LV capables d incorporer spécifiquement une enzyme, la bétalactamase (BLAM-LV) et de coder une forme tronquée du récepteur au facteur de croissance nerveuse (DELTA-NGFR), sont produits. Nos résultats montrent que les LV sont soumis à une restriction post-entrée forte dans les cellules hématopoïétiques, que ce soit dans des lymphocytes T immortalisés ou bien des CSH CD34+. Nous montrons également que cette inhibition post-entrée peut être partiellement saturée après une forte augmentation de la multiplicité d infection ou en présence d additifs de culture, comme la Vectofusin-1® ou laRetronectin®. De plus, nous avons montré lors de la transduction de CSPH avec des vecteurs BLAM-LV que la Vectofusin-1® agit sur l étape d entrée en augmentant l adhésion et la fusion entre les membranes virale et cellulaire. Cette technique représente donc un nouvel outil sensible et efficace pour étudier de façon concomitante l étape de fusion et le niveau de transduction dans les cellules cibles. A terme, ce travail permettra une meilleure compréhension de la biologie des LV mais pourra également conduire à l élaboration de protocoles de transduction lentivirale plus efficaces.Lentiviral vectors (LV) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking to precisely evaluate parameters that control the efficiency of transduction in relation with the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer (FRET)-based HIV-1 fusion assay to measure the entry of non-replicative recombinant LV in various cell types, including primary human hematopoietic stem and progenitor cells, and to quantify the level of transduction of he same initially-infected cells. The assay utilizes recombinant LV containing betalactamase (BLAM)-Vpr chimeric proteins (BLAM-LV) and encoding a truncated form of thelow affinity nerve growth factor receptor (DELTA-NGFR). This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing for instance the extent of lentiviral post-entry restrictions occuring in cells of hematopoietic origin. The assay also shows that transduction enhancers like Vectofusin®-1 or Retronectin® can partially relieve this post-entry block but their effects differ in the way to promote LV entry. Furthermore, our results show that Vectofusin®-1 acts at the entry step by promoting the adhesion and the fusion between lentiviral and cellular membranes. In conclusion, one such assay should be useful to study hematopoietic post-entry restrictions directed against LV and should allow improvements in various LV-based gene therapy protocols.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Concurrent measures of fusion and transduction efficiency of primary CD34+ cells with human immunodeficiency virus 1-based lentiviral vectors reveal different effects of transduction enhancers.: Concurrent study of lentiviral fusion/transduction

International audienceLentiviral vectors (LVs) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking for precisely evaluating parameters that control the efficiency of transduction in relation to the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer-based human immunodeficiency virus-1 fusion assay to measure the entry of nonreplicative recombinant LVs in various cell types, including primary human hematopoietic stem progenitor cells (HSPCs), and to quantify the level of transduction of the same initially infected cells. The assay utilizes recombinant LVs containing β-lactamase (BLAM)-Vpr chimeric proteins (BLAM-LVs) and encoding a truncated form of the low-affinity nerve growth factor receptor (ΔNGFR). After infection of target cells with BLAM-LVs, the vector entry rapidly leads to BLAM-Vpr release into the cytoplasm, which is measured by cleavage of a fluorescent substrate using flow cytometry. Parallel cultures of the same infected cells show transduction efficiency resulting from ΔNGFR expression. This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing, for instance, the postentry restrictions of LVs known to occur in cells of hematopoietic origin, especially human HSPCs. Furthermore, this BLAM-LV assay allowed us to evaluate the effect of cytokine prestimulation of HSPCs on the entry step of LVs. The assay also shows that transduction enhancers such as Vectofusin-1 or Retronectin can partially relieve the postentry block, but their effects differ in how they promote LV entry. In conclusion, one such assay should be useful to study hematopoietic postentry restrictions directed against LVs and therefore should allow improvements in various LV-based gene therapy protocols

HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion

AbstractWe recently described a sensitive and specific assay that detects the fusion of HIV-1 virions to a broad range of target cells, including primary CD4 cells. This assay involves the use of virions containing β-lactamase-Vpr (BlaM-Vpr) and the loading of target cells with CCF2, a fluorogenic substrate of β-lactamase. Since Vpr strongly associates with the viral core, uncoating of the viral particle might be required for effective cleavage of CCF2 by BlaM-Vpr. Here, we show that BlaM-Vpr within mature viral cores effectively cleaves CCF2, indicating that this assay measures virion fusion independently of uncoating. We also show that wildtype and Nef-deficient HIV-1 virions fuse with equivalent efficiency to HeLa-CD4 cells, SupT1 T cells, and primary CD4 T cells. Since Nef enhances cytoplasmic delivery of viral cores and increases viral infectivity, these findings indicate that Nef enhances an early post-fusion event in the multistep process of viral entry. Possible sites of Nef action include enlargement of the fusion pore, enhanced uncoating of viral particles, and more efficient passage of viral cores through the dense cortical actin network located immediately beneath the plasma membrane

Infectivity enhancement of different HIV-1-based lentiviral pseudotypes in presence of the cationic amphipathic peptide LAH4-L1.

International audienceLentiviral vectors (LVs) are promising delivery systems for gene therapy. To enhance the efficiency of target cell transduction by LVs, protocols often include the addition of culture additives. In this study, the cationic amphipathic peptide LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA), a DNA transfection agent, was evaluated for its capacity to improve LV infectivity in cell lines and primary cells. Results show that LAH4-L1 enhances infectivity of all LV pseudotypes tested, particularly GALVTR-LVs. More importantly, LAH4-L1 promotes the transduction of CD34+ hematopoietic stem cells with GALVTR-LVs as efficiently as Retronectin, a culture additive used in ex vivo clinical protocols involving LVs. The action of LAH4-L1 relies both on the GALVTR-LV adhesion and post-adhesion steps. LAH4-L1 represents a new and attractive transduction enhancer for hematopoietic gene therapy protocols