GBF1 est un facteur cellulaire requis pour la réplication du virus de l'hépatite C

Hepatitis C is a major cause of worldwide public health problems, worsened by the lack of vaccine and effective treatment. Therefore it is important to identify new potential therapeutic targets with an aim of developing more efficient treatments. For that, a better knowledge of the replicative cycle of hepatitis C virus (HCV) is essential. As observed for many other plus-strand RNA viruses, HCV induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous weblike structures induced by expression of HCV proteins in a non-replicative context, suggesting that GBF1 is probably not involved in the formation of HCV replication complexes, but rather in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication.L'hépatite C est un problème de santé publique majeur aggravé par l'efficacité limitée des thérapies actuelles et l'absence de vaccin. Il est donc important d'identifier de nouvelles cibles thérapeutiques potentielles et pour cela une meilleure connaissance du cycle infectieux du virus de l'hépatite C (VHC) est indispensable. Comme pour de nombreux autres virus à ARN de polarité positive, l'étape de réplication du VHC est associée à d'importants remaniements membranaires qui conduisent à la formation d'un réseau membranaire désigné membranous web et de structures membranaires plus petites associées au reticulum endoplasmique. Cependant, la formation des complexes de réplication du VHC, en association étroite avec ces membranes cellulaires modifiées reste énigmatique. Au niveau cellulaire, de petites GTPases sont connues pour réguler la dynamique des membranes. Nos travaux montrent que l'infection par le VHC est inhibée, de façon dose-dépendante, par la bréfeldine A (BFA), un inhibiteur spécifique de l'action de GTPases de la famille ARF. L'activation des petites GTPases ARF est médiée par des facteurs d'échange nucléotidiques. En utilisant des ARN interférants ciblant les trois facteurs d'échange nucléotidique sensibles à la BFA nous avons observé que seule la réduction de l'expression de l'un d'entre eux, appelé GBF1, inhibe l'infection par le VHC. Ces résultats ont été confirmés par l'utilisation d'un inhibiteur chimique spécifique. D'autre part, la surexpression d'un mutant de GBF1 résistant à la BFA permet de restaurer l'infection par le VHC en présence de BFA. Par ailleurs, des analyses en immunofluorescence et microscopie électronique réalisées dans un contexte non réplicatif montrent que la BFA n'inhibe pas les réarrangements membranaires associés à la mise en place du membranous web. Collectivement nos résultats suggèrent que GBF1 est impliqué dans l'activité des complexes de réplication du VHC et notre travail établit un lien entre la voie de sécrétion de la cellule hôte et la réplication de l'ARN du VHC

GBF1 est un facteur cellulaire requis pour la réplication du virus de l'hépatite C

L hépatite C est un problème de santé publique majeur aggravé par l efficacité limitée des thérapies actuelles et l absence de vaccin. Il est donc important d identifier de nouvelles cibles thérapeutiques potentielles et pour cela une meilleure connaissance du cycle infectieux du virus de l hépatite C (VHC) est indispensable. Comme pour de nombreux autres virus à ARN de polarité positive, l étape de réplication du VHC est associée à d importants remaniements membranaires qui conduisent à la formation d un réseau membranaire désigné membranous web et de structures membranaires plus petites associées au reticulum endoplasmique. Cependant, la formation des complexes de réplication du VHC, en association étroite avec ces membranes cellulaires modifiées reste énigmatique. Au niveau cellulaire, de petites GTPases sont connues pour réguler la dynamique des membranes. Nos travaux montrent que l infection par le VHC est inhibée, de façon dose-dépendante, par la bréfeldine A (BFA), un inhibiteur spécifique de l action de GTPases de la famille ARF. L activation des petites GTPases ARF est médiée par des facteurs d échange nucléotidiques. En utilisant des ARN interférants ciblant les trois facteurs d échange nucléotidique sensibles à la BFA nous avons observé que seule la réduction de l expression de l un d entre eux, appelé GBF1, inhibe l infection par le VHC. Ces résultats ont été confirmés par l utilisation d un inhibiteur chimique spécifique. D autre part, la surexpression d un mutant de GBF1 résistant à la BFA permet de restaurer l infection par le VHC en présence de BFA. Par ailleurs, des analyses en immunofluorescence et microscopie électronique réalisées dans un contexte non réplicatif montrent que la BFA n inhibe pas les réarrangements membranaires associés à la mise en place du membranous web. Collectivement nos résultats suggèrent que GBF1 est impliqué dans l activité des complexes de réplication du VHC et notre travail établit un lien entre la voie de sécrétion de la cellule hôte et la réplication de l ARN du VHC.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Hepatitis C virus replication and Golgi function in brefeldin a-resistant hepatoma-derived cells.

Recent reports indicate that the replication of hepatitis C virus (HCV) depends on the GBF1-Arf1-COP-I pathway. We generated Huh-7-derived cell lines resistant to brefeldin A (BFA), which is an inhibitor of this pathway. The resistant cell lines could be sorted into two phenotypes regarding BFA-induced toxicity, inhibition of albumin secretion, and inhibition of HCV infection. Two cell lines were more than 100 times more resistant to BFA than the parental Huh-7 cells in these 3 assays. This resistant phenotype was correlated with the presence of a point mutation in the Sec7 domain of GBF1, which is known to impair the binding of BFA. Surprisingly, the morphology of the cis-Golgi of these cells remained sensitive to BFA at concentrations of the drug that allowed albumin secretion, indicating a dichotomy between the phenotypes of secretion and Golgi morphology. Cells of the second group were about 10 times more resistant than parental Huh-7 cells to the BFA-induced toxicity. The EC50 for albumin secretion was only 1.5-1.8 fold higher in these cells than in Huh-7 cells. However their level of secretion in the presence of inhibitory doses of BFA was 5 to 15 times higher. Despite this partially effective secretory pathway in the presence of BFA, the HCV infection was almost as sensitive to BFA as in Huh-7 cells. This suggests that the function of GBF1 in HCV replication does not simply reflect its role of regulator of the secretory pathway of the host cell. Thus, our results confirm the involvement of GBF1 in HCV replication, and suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication

Identification of class II ADP-ribosylation factors as cellular factors required for hepatitis C virus replication

International audienceGBF1 is a host factor required for hepatitis C virus (HCV) replication. GBF1 functions as a guanine nucleotide exchange factor for G‐proteins of the Arf family, which regulate membrane dynamics in the early secretory pathway and the metabolism of cytoplasmic lipid droplets. Here we established that the Arf‐guanine nucleotide exchange factor activity of GBF1 is critical for its function in HCV replication, indicating that it promotes viral replication by activating one or more Arf family members. Arf involvement was confirmed with the use of two dominant negative Arf1 mutants. However, siRNA‐mediated depletion of Arf1, Arf3 (class I Arfs), Arf4 or Arf5 (class II Arfs), which potentially interact with GBF1, did not significantly inhibit HCV infection. In contrast, the simultaneous depletion of both Arf4 and Arf5, but not of any other Arf pair, imposed a significant inhibition of HCV infection. Interestingly, the simultaneous depletion of both Arf4 and Arf5 had no impact on the activity of the secretory pathway and induced a compaction of the Golgi and an accumulation of lipid droplets. A similar phenotype of lipid droplet accumulation was also observed when GBF1 was inhibited by brefeldin A. In contrast, the simultaneous depletion of both Arf1 and Arf4 resulted in secretion inhibition and Golgi scattering, two actions reminiscent of GBF1 inhibition. We conclude that GBF1 could regulate different metabolic pathways through the activation of different pairs of Arf proteins

Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein

International audienceGBF1 has emerged as a host factor required for the genome replication of RNA viruses of different families. During the hepatitis C virus (HCV) life cycle, GBF1 performs a critical function at the onset of genome replication but is dispensable when the replication is established. To better understand how GBF1 regulates HCV infection, we have looked for interactions between GBF1 and HCV proteins. NS3 was found to interact with GBF1 in yeast two-hybrid, coimmunoprecipitation, and proximity ligation assays and to interfere with GBF1 function and alter GBF1 intracellular localization in cells expressing NS3. The interaction was mapped to the Sec7 domain of GBF1 and the protease domain of NS3. A reverse yeast two-hybrid screen to identify mutations altering NS3-GBF1 interaction yielded an NS3 mutant (N77D, Con1 strain) that is nonreplicative despite conserved protease activity and does not interact with GBF1. The mutated residue is exposed at the surface of NS3, suggesting it is part of the domain of NS3 that interacts with GBF1. The corresponding mutation in strain JFH-1 (S77D) produces a similar phenotype. Our results provide evidence for an interaction between NS3 and GBF1 and suggest that an alteration of this interaction is detrimental to HCV genome replication.IMPORTANCE: Single-stranded, positive-sense RNA viruses rely to a significant extent on host factors to achieve the replication of their genome. GBF1 is such a cellular protein that is required for the replication of several RNA viruses, but its mechanism of action during viral infections is not yet defined. In this study, we investigated potential interactions that GBF1 might engage in with proteins of HCV, a GBF1-dependent virus. We found that GBF1 interacts with NS3, a nonstructural protein involved in HCV genome replication, and our results suggest that this interaction is important for GBF1 function during HCV replication. Interestingly, GBF1 interaction with HCV appears different from its interaction with enteroviruses, another group of GBF1-dependent RNA viruses, in keeping with the fact that HCV and enteroviruses use different functions of GBF1

BFA sensitivity of the cis-Golgi of Huh-7, R1, R2 and MDCK cells.

<p>Cells were treated for 30 minutes with increasing concentrations of BFA, fixed and processed for the immunofluorescent detection of GM130. For each condition, approximately 100 cells were scored for their cis-Golgi morphology, as either intact or scattered. For each cell line, the percentages of cells with intact cis-Golgi morphology were plotted against BFA concentrations.</p

Mutation detected in GBF1 of R1 and R2 cells.

<p>(A) A fraction of the electrophoregrams corresponding to the sequence of GBF1 from the indicated cell lines is presented. The nucleotide and amino-acid sequences are indicated. The position of the mutation is indicated by an arrow. (B) Huh-7 cells were transfected with expression plasmids for GBF1-M832L, GBF1 inactive mutant E794K, or YFP. Transfected cells were submitted to a cell viability assay, as explained in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074491#pone-0074491-g001" target="_blank">figure 1</a>. Results were expressed as percentages of the values obtained with no BFA. Error bars represent the SEM of 3 independent experiments performed in triplicates. (C) Transfected cells were seeded in 12-well plates, and cultured in the presence of BFA for 24 h. The amounts of human serum albumin (HSA) in the conditioned culture media and in cell lysates were quantified with an ELISA assay and expressed as percentages of HSA secretion. Error bars represent the SEM of 3 independent experiments performed in duplicates.</p

Viability of BFA-resistant cells.

<p>Sub-confluent cells of the indicated cell lines were cultured in 96-well plates in the presence of the indicated concentrations of BFA or of 0.2% ethanol (BFA stock solvent) for 24 h. Viability was assessed using an MTS assay. The absorbance of the ethanol-treated sample is expressed as 100%. Results were expressed as percentages of the values obtained with no BFA. Error bars represent the SEM of 2 independent experiments performed in triplicates.</p

Mutations found in the coding sequence of GBF1 in Huh-7 cells.

a<p>Genbank NM_001199379.1.</p

Half maximal effective concentrations of BFA effects on the cis-Golgi morphology and HSA secretion.

<p>Half maximal effective concentrations of BFA effects on the cis-Golgi morphology and HSA secretion.</p