Etude des mécanismes dépendants de GBF1 et impliqués dans la réplication du virus de l'hépatite C

The hepatitis C virus (HCV) infection progresses in most of the cases into a chronic hepatitis and can lead to cirrhosis or hepatocellular carcinoma. Despite the recent improvement of hepatitis C treatments, which inhibit or even block the progress of this infection into a chronic stage, a vaccine still not available and the worldwide distribution of the disease makes the hepatitis C a major public health problem. Most of the available treatments target viral proteins. However many mechanisms of the HCV life cycle remain unclear.As for many positive RNA viruses, HCV replication occurs in reorganized cellular membranes. These membrane rearrangements are closely linked to the early secretory pathway of the cell. It has been shown that GBF1, an exchange factor of small G proteins of the Arf family that regulates the membrane dynamics in the secretory pathway, is required for HCV replication. GBF1 inhibition by brefeldin A (BFA) inhibits the secretion of newly synthesized proteins and also inhibits HCV replication. To investigate the role of GBF1 in HCV infection, we isolated cell lines resistant to BFA. Two of these cell lines were 100 times more resistant than the parental cells to BFA-induced apoptosis, inhibition of proteins secretion and inhibition of HCV infection. This resistance was due to a point mutation in the catalytic sec7 domain of GBF1 of these cells. Another group of resistant cells was showing a partial resistance to the inhibition of proteins secretion while maintaining their sensitivity to the inhibition of HCV infection in the same conditions. These results suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication. Using GBF1 deletion mutants we showed that the catalytic activity of the sec7 domain of GBF1 is required for HCV infection. This suggests that the function of GBF1 during HCV replication is mediated by Arf activation. The involvement of Arf was confirmed with the overexpression of restricted mutants of Arf1 and by the inhibition of ArfGAP1, another regulator of Arf function. We then tested the possible involvement of different Arfs (Arf1, 3, 4 and 5) in HCV infection. It has been reported that Arfs have redundant functions. The results confirm the involvement of Arf1 and indicate that all the other BFA-sensitive Arfs (Arf3, Arf4 and Arf5) are also involved in HCV infection. The combined knockdown of Arfs strongly inhibited HCV replication, showing that the Arf proteins are working together in HCV replication probably by activating several host factors required for the virus life cycle.The study of cellular factors required for HCV infection is crucial to better understand the interaction of the virus with the host cell and thus the whole HCV life cycle. This could help to develop new therapies targeting the host cell, regardless of viral genotypes and reducing the risk of emergence of new resistant forms.L’infection par le virus de l’hépatite C (HCV) évolue dans la plupart des cas en hépatite chronique et peut conduire à une cirrhose ou un carcinome hépatocellulaire. Malgré les grandes avancées dans le traitement de l’hépatite C qui permettent d’inhiber ou même de bloquer l’évolution de cette infection vers la chronicité, l’absence de vaccin ainsi que sa répartition sur la surface du globe nous permet de classer cette pathologie en problème majeur de santé publique. La majorité des traitements actuels ciblent les protéines virales et leur fonction. Cependant un grand nombre de mécanismes du cycle viral de HCV reste à élucider.Comme pour la grande majorité des virus à ARN de polarité positive, la réplication de HCV a lieu dans des membranes cellulaires modifiées. Le remaniement de ces membranes est en lien étroit avec la voie de sécrétion précoce de la cellule. Il a été montré que GBF1, un facteur d’échange nucléotidique des protéines G de la famille Arf qui régulent la dynamique membranaire, est un facteur nécessaire à la réplication de HCV. L’inhibition de GBF1 par la bréfeldine A (BFA) inhibe la voie de sécrétion des protéines cellulaires néosynthétisées et inhibe aussi la réplication de HCV. Pour étudier le rôle de GBF1 pendant l’infection nous avons établi des lignées résistantes à la BFA. Deux de ces lignées étaient 100 fois plus résistantes que les lignées parentales à l’apoptose induite par la BFA, à l’inhibition de la sécrétion des protéines et à l’inhibition de l’infection par HCV. Ce phénotype était dû à une mutation ponctuelle dans le domaine catalytique sec7 de GBF1 de ces lignées. Un autre groupe de lignées était partiellement résistantes à l’inhibition de la sécrétion des protéines par la BFA tout en conservant un niveau d’infection proche de celui des lignées parentales dans les mêmes conditions. Ces résultats suggèrent que la fonction de GBF1 pendant l’infection HCV ne serait pas réduite à la régulation de la voie de sécrétion, évoquant ainsi un rôle additionnel de GBF1 nécessaire pour la réplication de HCV.Par ailleurs, nous avons pu montrer à l’aide des mutants de délétion de la protéine GBF1, que l’activité catalytique du domaine sec7 était nécessaire. Ceci suggère l’implication d’une protéine de la famille Arf dans l’activation de l’infection HCV via GBF1. L’implication de Arf dans l’infection HCV a été confirmée par la surexpression de dominants négatifs de la protéine Arf1 et par l’inhibition de l’activité de l’ArfGAP1 (régulateur des Arf) par l’inhibiteur spécifique QS11.Nous avons ensuite testé l’implication des différents Arf sensibles à l’inhibition par la BFA (Arf1, 3 ,4 et 5), dans l’infection HCV à l’aide de si-RNA. Il a été montré que ces protéines Arf possèdent des fonctions redondantes. Nos résultats confirment l’implication de Arf1 et indiquent que les 3 autres protéines sont aussi impliquées dans l’infection HCV. D’une manière intéressante, la déplétion combinée des Arf inhibe fortement l’infection HCV suggérant ainsi un rôle essentiel de certaines protéines Arf, probablement en activant des facteurs cellulaires nécessaires à l’étape de réplication. L’étude des facteurs cellulaires impliqués dans l’infection HCV permet de mieux comprendre l’étape de réplication et par conséquent le cycle viral de HCV. Par ailleurs, l’étude de ces facteurs pourrait permettre le développement éventuel de stratégies antivirales ciblant des facteurs de la cellule hépatique indépendamment du génotype viral, limitant ainsi le risque d’émergence de variants résistants au traitement

Investigation of GBF1-dependent mechanisms involved in hepatitis C virus replication

L’infection par le virus de l’hépatite C (HCV) évolue dans la plupart des cas en hépatite chronique et peut conduire à une cirrhose ou un carcinome hépatocellulaire. Malgré les grandes avancées dans le traitement de l’hépatite C qui permettent d’inhiber ou même de bloquer l’évolution de cette infection vers la chronicité, l’absence de vaccin ainsi que sa répartition sur la surface du globe nous permet de classer cette pathologie en problème majeur de santé publique. La majorité des traitements actuels ciblent les protéines virales et leur fonction. Cependant un grand nombre de mécanismes du cycle viral de HCV reste à élucider.Comme pour la grande majorité des virus à ARN de polarité positive, la réplication de HCV a lieu dans des membranes cellulaires modifiées. Le remaniement de ces membranes est en lien étroit avec la voie de sécrétion précoce de la cellule. Il a été montré que GBF1, un facteur d’échange nucléotidique des protéines G de la famille Arf qui régulent la dynamique membranaire, est un facteur nécessaire à la réplication de HCV. L’inhibition de GBF1 par la bréfeldine A (BFA) inhibe la voie de sécrétion des protéines cellulaires néosynthétisées et inhibe aussi la réplication de HCV. Pour étudier le rôle de GBF1 pendant l’infection nous avons établi des lignées résistantes à la BFA. Deux de ces lignées étaient 100 fois plus résistantes que les lignées parentales à l’apoptose induite par la BFA, à l’inhibition de la sécrétion des protéines et à l’inhibition de l’infection par HCV. Ce phénotype était dû à une mutation ponctuelle dans le domaine catalytique sec7 de GBF1 de ces lignées. Un autre groupe de lignées était partiellement résistantes à l’inhibition de la sécrétion des protéines par la BFA tout en conservant un niveau d’infection proche de celui des lignées parentales dans les mêmes conditions. Ces résultats suggèrent que la fonction de GBF1 pendant l’infection HCV ne serait pas réduite à la régulation de la voie de sécrétion, évoquant ainsi un rôle additionnel de GBF1 nécessaire pour la réplication de HCV.Par ailleurs, nous avons pu montrer à l’aide des mutants de délétion de la protéine GBF1, que l’activité catalytique du domaine sec7 était nécessaire. Ceci suggère l’implication d’une protéine de la famille Arf dans l’activation de l’infection HCV via GBF1. L’implication de Arf dans l’infection HCV a été confirmée par la surexpression de dominants négatifs de la protéine Arf1 et par l’inhibition de l’activité de l’ArfGAP1 (régulateur des Arf) par l’inhibiteur spécifique QS11.Nous avons ensuite testé l’implication des différents Arf sensibles à l’inhibition par la BFA (Arf1, 3 ,4 et 5), dans l’infection HCV à l’aide de si-RNA. Il a été montré que ces protéines Arf possèdent des fonctions redondantes. Nos résultats confirment l’implication de Arf1 et indiquent que les 3 autres protéines sont aussi impliquées dans l’infection HCV. D’une manière intéressante, la déplétion combinée des Arf inhibe fortement l’infection HCV suggérant ainsi un rôle essentiel de certaines protéines Arf, probablement en activant des facteurs cellulaires nécessaires à l’étape de réplication. L’étude des facteurs cellulaires impliqués dans l’infection HCV permet de mieux comprendre l’étape de réplication et par conséquent le cycle viral de HCV. Par ailleurs, l’étude de ces facteurs pourrait permettre le développement éventuel de stratégies antivirales ciblant des facteurs de la cellule hépatique indépendamment du génotype viral, limitant ainsi le risque d’émergence de variants résistants au traitement.The hepatitis C virus (HCV) infection progresses in most of the cases into a chronic hepatitis and can lead to cirrhosis or hepatocellular carcinoma. Despite the recent improvement of hepatitis C treatments, which inhibit or even block the progress of this infection into a chronic stage, a vaccine still not available and the worldwide distribution of the disease makes the hepatitis C a major public health problem. Most of the available treatments target viral proteins. However many mechanisms of the HCV life cycle remain unclear.As for many positive RNA viruses, HCV replication occurs in reorganized cellular membranes. These membrane rearrangements are closely linked to the early secretory pathway of the cell. It has been shown that GBF1, an exchange factor of small G proteins of the Arf family that regulates the membrane dynamics in the secretory pathway, is required for HCV replication. GBF1 inhibition by brefeldin A (BFA) inhibits the secretion of newly synthesized proteins and also inhibits HCV replication. To investigate the role of GBF1 in HCV infection, we isolated cell lines resistant to BFA. Two of these cell lines were 100 times more resistant than the parental cells to BFA-induced apoptosis, inhibition of proteins secretion and inhibition of HCV infection. This resistance was due to a point mutation in the catalytic sec7 domain of GBF1 of these cells. Another group of resistant cells was showing a partial resistance to the inhibition of proteins secretion while maintaining their sensitivity to the inhibition of HCV infection in the same conditions. These results suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication. Using GBF1 deletion mutants we showed that the catalytic activity of the sec7 domain of GBF1 is required for HCV infection. This suggests that the function of GBF1 during HCV replication is mediated by Arf activation. The involvement of Arf was confirmed with the overexpression of restricted mutants of Arf1 and by the inhibition of ArfGAP1, another regulator of Arf function. We then tested the possible involvement of different Arfs (Arf1, 3, 4 and 5) in HCV infection. It has been reported that Arfs have redundant functions. The results confirm the involvement of Arf1 and indicate that all the other BFA-sensitive Arfs (Arf3, Arf4 and Arf5) are also involved in HCV infection. The combined knockdown of Arfs strongly inhibited HCV replication, showing that the Arf proteins are working together in HCV replication probably by activating several host factors required for the virus life cycle.The study of cellular factors required for HCV infection is crucial to better understand the interaction of the virus with the host cell and thus the whole HCV life cycle. This could help to develop new therapies targeting the host cell, regardless of viral genotypes and reducing the risk of emergence of new resistant forms

Hepatitis C Virus Life Cycle and Lipid Metabolism

Hepatitis C Virus (HCV) infects over 150 million people worldwide. In most cases HCV infection becomes chronic, causing liver disease ranging from fibrosis to cirrhosis and hepatocellular carcinoma. HCV affects the cholesterol homeostasis and at the molecular level, every step of the virus life cycle is intimately connected to lipid metabolism. In this review, we present an update on the lipids and apolipoproteins that are involved in the HCV infectious cycle steps: entry, replication and assembly. Moreover, the result of the assembly process is a lipoviroparticle, which represents a peculiarity of hepatitis C virion. This review illustrates an example of an intricate virus-host interaction governed by lipid metabolism

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

Investigation of the role of GBF1 in the replication of positive-sense single-stranded RNA viruses

International audienceGBF1 has emerged as a host factor required for the replication of positive-sense single-stranded RNA viruses of different families, but its mechanism of action is still unknown. GBF1 is a guanine nucleotide exchange factor for Arf family members. Recently, we identified Arf4 and Arf5 (class II Arfs) as host factors required for the replication of hepatitis C virus (HCV), a GBF1-dependent virus. To assess whether a GBF1/class II Arf pathway is conserved among positive-sense single-stranded RNA viruses, we investigated yellow fever virus (YFV), Sindbis virus (SINV), coxsackievirus B4 (CVB4) and human coronavirus 229E (HCoV-229E). We found that GBF1 is involved in the replication of these viruses. However, using siRNA or CRISPR-Cas9 technologies, it was seen that the depletion of Arf1, Arf3, Arf4 or Arf5 had no impact on viral replication. In contrast, the depletion of Arf pairs suggested that class II Arfs could be involved in HCoV-229E, YFV and SINV infection, as for HCV, but not in CVB4 infection. In addition, another Arf pair, Arf1 and Arf4, appears to be essential for YFV and SINV infection, but not for infection by other viruses. Finally, CVB4 infection was not inhibited by any combination of Arf depletion. We conclude that the mechanism of action of GBF1 in viral replication appears not to be conserved, and that a subset of positive-sense single-stranded RNA viruses from different families might require class II Arfs for their replication

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

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