Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly.: HCV core protein cleavage by SPP and viral assembly

International audienceHepatitis C virus (HCV) core protein, expressed with a Semliki Forest virus replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. This model was used to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. Several mutants were designed as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. These data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly

Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection.: HCV-induced membrane rearrangements

International audienceLike most positive-strand RNA viruses, hepatitis C virus (HCV) forms a membrane-associated replication complex consisting of replicating RNA, viral and host proteins anchored to altered cell membranes. We used a combination of qualitative and quantitative electron microscopy (EM), immuno-EM, and the 3D reconstruction of serial EM sections to analyze the host cell membrane alterations induced by HCV. Three different types of membrane alteration were observed: vesicles in clusters (ViCs), contiguous vesicles (CVs), and double-membrane vesicles (DMVs). The main ultrastructural change observed early in infection was the formation of a network of CVs surrounding the lipid droplets. Later stages in the infectious cycle were characterized by a large increase in the number of DMVs, which may be derived from the CVs. These DMVs are thought to constitute the membranous structures harboring the viral replication complexes in which viral replication is firmly and permanently established and to protect the virus against double-stranded RNA-triggered host antiviral responses

Antigenic Properties of Recombinant Envelope Glycoproteins Derived from T-Cell-Line-Adapted Isolates or Primary Human Immunodeficiency Virus Isolates and Their Relationship to Immunogenicity

AbstractThe native envelope glycoproteins of primary HIV-1 virions have weaker antigenicity than do T-cell laboratory-adapted (TCLA) viruses. These antigenic properties require further evaluation if recombinant envelope glycoproteins are produced as part of a vaccine strategy. In this study, we compared the antigenicity of recombinant envelope glycoproteins derived from three primary isolates (PI) (HIV-1BX08, HIV-1CHA, and HIV-1133) and two TCLA viruses (HIV-1HXB2 and HIV-1MN) produced using the Semliki Forest virus (SFV) system. This analysis was performed by radioimmunoprecipitation assays and flow cytometry. The results suggest that the SFV produces envelope glycoproteins with features in common with the envelopes found in naturally occurring virions. In particular, the PI envelopes had weak heterogeneous antigenic properties. However, the cytometric analysis also showed that there was less envelope glycoprotein on the cell surface for the PI envelopes than for those of TCLA viruses, suggesting differences in their intracellular trafficking. The immunogenic properties of the various envelope glycoproteins were evaluated in mice using recombinant SFV particles as vaccine vectors. The PI envelopes were less immunogenic than the TCLA envelopes, probably due to both their low antigenicity and cell surface expression level. Thus, it may be difficult to design an effective vaccine based on native recombinant PI envelopes

Suppression of LPS-induced inflammatory responses in macrophages infected with Leishmania

<p>Abstract</p> <p>Background</p> <p>Chronic inflammation activated by macrophage innate pathogen recognition receptors such as TLR4 can lead to a range of inflammatory diseases, including atherosclerosis, Crohn's disease, arthritis and cancer. Unlike many microbes, the kinetoplastid protozoan pathogen <it>Leishmania </it>has been shown to avoid and even actively suppress host inflammatory cytokine responses, such as LPS-induced IL-12 production. The nature and scope of <it>Leishmania</it>-mediated inflammatory cytokine suppression, however, is not well characterized. Advancing our knowledge of such microbe-mediated cytokine suppression may provide new avenues for therapeutic intervention in inflammatory disease.</p> <p>Methods</p> <p>We explored the kinetics of a range of cytokine and chemokine responses in primary murine macrophages stimulated with LPS in the presence versus absence of two clinically distinct species of <it>Leishmania </it>using sensitive multiplex cytokine analyses. To confirm that these effects were parasite-specific, we compared the effects of <it>Leishmania </it>uptake on LPS-induced cytokine expression with uptake of inert latex beads.</p> <p>Results</p> <p>Whilst <it>Leishmania </it>uptake alone did not induce significant levels of any cytokine analysed in this study, <it>Leishmania </it>uptake in the presence of LPS caused parasite-specific suppression of certain LPS-induced pro-inflammatory cytokines, including IL-12, IL-17 and IL-6. Interestingly, <it>L. amazonensis </it>was generally more suppressive than <it>L. major</it>. We also found that other LPS-induced proinflammatory cytokines, such as IL-1α, TNF-α and the chemokines MIP-1α and MCP-1 and also the anti-inflammatory cytokine IL-10, were augmented during <it>Leishmania </it>uptake, in a parasite-specific manner.</p> <p>Conclusions</p> <p>During uptake by macrophages, <it>Leishmania </it>evades the activation of a broad range of cytokines and chemokines. Further, in the presence of a strong inflammatory stimulus, <it>Leishmania </it>suppresses certain proinflammatory cytokine responses in a parasite-specific manner, however it augments the production of other proinflammatory cytokines. Our findings highlight the complexity of inflammatory cytokine signalling regulation in the context of the macrophage and <it>Leishmania </it>interaction and confirm the utility of the <it>Leishmania</it>/macrophage infection model as an experimental system for further studies of inflammatory regulation. Such studies may advance the development of therapies against inflammatory disease.</p

A Standard Siren Measurement of the Hubble Constant from GW170817 without the Electromagnetic Counterpart

We perform a statistical standard siren analysis of GW170817. Our analysis does not utilize knowledge of NGC 4993 as the unique host galaxy of the optical counterpart to GW170817. Instead, we consider each galaxy within the GW170817 localization region as a potential host; combining the redshifts from all of the galaxies with the distance estimate from GW170817 provides an estimate of the Hubble constant, H 0. Considering all galaxies brighter than $0.626{L}_{B}^{\star }$ as equally likely to host a binary neutron star merger, we find ${H}_{0}={77}_{-18}^{+37}$ km s−1 Mpc−1 (maximum a posteriori and 68.3% highest density posterior interval; assuming a flat H 0 prior in the range $\left[10,220\right]$ km s−1 Mpc−1). We explore the dependence of our results on the thresholds by which galaxies are included in our sample, and we show that weighting the host galaxies by stellar mass or star formation rate provides entirely consistent results with potentially tighter constraints. By applying the method to simulated gravitational-wave events and a realistic galaxy catalog we show that, because of the small localization volume, this statistical standard siren analysis of GW170817 provides an unusually informative (top 10%) constraint. Under optimistic assumptions for galaxy completeness and redshift uncertainty, we find that dark binary neutron star measurements of H 0 will converge as $40 \% /\sqrt{(N)}$, where N is the number of sources. While these statistical estimates are inferior to the value from the counterpart standard siren measurement utilizing NGC 4993 as the unique host, ${H}_{0}={76}_{-13}^{+19}$ km s−1 Mpc−1 (determined from the same publicly available data), our analysis is a proof-of-principle demonstration of the statistical approach first proposed by Bernard Schutz over 30 yr ago

Les Arboviroses (conseils et prévention pour les voyageurs)

TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Les Virus grippaux (un problème de santé publique nécessitant le développement de nouveaux modèles animaux d'infection pour la mise au point de vaccins)

Les épidémies de grippe sont une cause majeure de morbidité et de mortalité dans le monde. Il y a ainsi chaque année 3 à 5 millions de cas de grippe grave et 0,25 à 0,5 millions de décès. En France, la grippe touche annuellement 2 à 8 millions de personnes et provoque entre 1500 et 2000 décès, essentiellement chez les personnes agées ou fragilisées. Les virus Influenza A et B (famille de Orthomyxoviridae) sont les agents étiologiques responsables de la grippe. Seuls les virus Influenza A sont à l'origine d'épidémies exceptionnelles par leur extension géographique et/ou leur gravité. La période d'incubation de la maladie varie de 1 à 3 jours. Les symptomes apparaissent brutalement avec principalement de la fièvre, une asthénie, des courbatures et des céphalées. Ces symptomes disparaissent habituellement en une à deux semaines. Des complications graves dues aux virus eux-mêmes ou aux surinfections bactériennes (pneumonie) peuvent survenir. Les vaccins restent le moyen le plus efficace de prévenir la morbidité et la mortalité dues à la grippe dans les populations à risque. Leur mise au point repose sur le développement de modèles animaux pertinents en particulier pour l'infection par le virus Influenza A. La souris étant un bon modèle pour les souches A/H1N1, nous présentons dans ce travail de thèse une étude expérimentale visant à déterminer la dose virale nécessaire pour obtenir chez cet animal une physiopathologie similaire à celle observée chez l'homme.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Etude de la morphogénèse de pseudo-virions du virus de l'hépatite C

Pour contourner l'abscence de système de culture in vitro du virus de l'hépatite C (VHC), nous avons developpé une stratégie visant à l'obtention de pseudo-virions du VHC en exprimant les protéines structurales virales, à savoir la protéine de capside et les deux glycoprotéines d'enveloppe E1 et E2, en cellules de mammifères. Ainsi, nous avons montré que l'expression de cette polyprotéine partielle virale, à l'aide d'un vecteur dérivé du virus de la forêt de Semliki, conduisait à la morphogénèse de pseudo-virions structurés du VHC. Cependant, les images de microscopie électronique ont montré que ce bourgeonnement, visualisé au sein de la membrane du réticulum endoplasmique(RE), semblait abortif. En dépit de cette absence de sécrétion, nous avons pu démontrer que la protéine de capside seule, était capable d'initier le bourgeonnement en pseudo-virions vers le RE, et que la mutation d'un seul acide aminé dans cette protéine abolissait complètement ce processus de bourgeonnement.TOURS-BU Sciences Pharmacie (372612104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Etude du trafic intracellulaire des glycoprotéines d'enveloppe d'isolats primaires du virus de l'immunodéficience humaine de type 1 et de son impact sur l'assemblage viral

Les glycoprotéines d'enveloppe (Env) de VIH-1 se caractérisent par une variabilité génétique qui peut toucher les motifs impliqués dans la régulation de leur trafic intracellulaire. Nous avons ainsi mis en évidence, pour certaines Env d'isolats primaires, un polymorphisme dans ces motifs. Nous avons pu montrer que ce polymorphisme naturel affectait la distribution intracellulaire des Env. Cette modification du trafic intracellulaire perturbe l'assemblage viral en diminuant l'incorporation des Env à la surface des virions, et de ce fait les capacités réplicatives des virus produits. Cependant, il semble que, dans les Env. de VIH-1 primaires, des déterminants additionnels, puissent modifier le trafic intracellulaire de ces protéines. Cette modification de trafic pourrait, par ailleurs, contribuer à l'échappement du virus au système immunitaire. Ces travaux amènent de nouveaux éléments sur la compréhension des moyens adoptés in vivo par le VIH-1 pour s'assurer d'une propagation optimale.The envelope glycoprotein (Env.) of HIV-1 is characterized by an important polymorphism that can affect motifs involved in the regulation of the intracellular trafficking. her, we investigated four envelope genes with natural polymorphism within these motifs. We showed that this polymorphism might influence the intracellular distribution of Env. This modification affects viral assembly by diminution of Env incorporation into virions and, thus, viral replication capacity. Furthermore, it seems that additional determinants regulate intacellular trafficking of primary Env. This traffic's modification could in part contribute to viral evade from immune system.These work bring new insight in the understanding of viral life and its capacity to insure optimal propagation in vivo.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF