Hepatitis C Virus Regulates its Replication by Maturing miR-122 Through Akt-Dependent Phosphorylation of KSRP

AbstractThe liver-specific micro-RNA-122 (miR-122) is required for the replication of hepatitis C virus (HCV). The direct interaction between miR-122 and the 5’ untranslated region of the HCV genome promotes viral replication and protects HCV RNA from degradation. Because HCV RNA is its own substrate for replication, infected cells are submitted to the sequestration of increasing levels of miR-122 and to global de-repression of host miR-122 mRNA targets. Whether and how HCV regulates miR-122 maturation to create an environment favorable to its replication remains unexplored. We discovered that Akt-dependent phosphorylation of KSRP host protein at Serine residue 193 is essential for miR-122 maturation in hepatocytes. Moreover, we showed the existence of a reciprocal regulation loop where HCV replication can modulate the proviral effect mediated by KSRP-dependent maturation of miR-122. These data support a mechanism by which HCV regulates the expression of miR-122 by hijacking KSRP, thereby fueling its own replication.

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Using Pharmacokinetic and Viral Kinetic Modeling To Estimate the Antiviral Effectiveness of Telaprevir, Boceprevir, and Pegylated Interferon during Triple Therapy in Treatment-Experienced Hepatitis C Virus-Infected Cirrhotic Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02611-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5332-5341 ◽

Cited By ~ 8

Author(s):

Cédric Laouénan ◽

Patrick Marcellin ◽

Martine Lapalus ◽

Feryel Khelifa-Mouri ◽

Nathalie Boyer ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Triple Therapy ◽

Pegylated Interferon ◽

Hcv Rna ◽

Second Phase ◽

Compensated Cirrhosis ◽

Viral Kinetic ◽

Significant Difference ◽

Infected Cells

ABSTRACTTriple therapy combining a protease inhibitor (PI) (telaprevir or boceprevir), pegylated interferon (PEG-IFN), and ribavirin (RBV) has dramatically increased the chance of eradicating hepatitis C virus (HCV). However, the efficacy of this treatment remains suboptimal in cirrhotic treatment-experienced patients. Here, we aimed to better understand the origin of this impaired response by estimating the antiviral effectiveness of each drug. Fifteen HCV genotype 1-infected patients with compensated cirrhosis, who were nonresponders to prior PEG-IFN/RBV therapy, were enrolled in a nonrandomized study. HCV RNA and concentrations of PIs, PEG-IFN, and RBV were frequently assessed in the first 12 weeks of treatment and were analyzed using a pharmacokinetic/viral kinetic model. The two PIs achieved similar levels of molar concentrations (P= 0.5), but there was a significant difference in the 50% effective concentrations (EC50) (P= 0.008), leading to greater effectiveness for telaprevir than for boceprevir in blocking viral production (99.8% versus 99.0%, respectively,P= 0.002). In all patients, the antiviral effectiveness of PEG-IFN was modest (43.4%), and there was no significant contribution of RBV exposure to the total antiviral effectiveness. The second phase of viral decline, which is attributed to the loss rate of infected cells, was slow (0.19 day−1) and was higher in patients who subsequently eradicated HCV (P= 0.03). The two PIs achieved high levels of antiviral effectiveness. However, the suboptimal antiviral effectiveness of PEG-IFN/RBV and the low loss of infected cells suggest that a longer treatment duration might be needed in cirrhotic treatment-experienced patients and that a future IFN-free regimen may be particularly beneficial in these patients.

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Visualization of Double-Stranded RNA in Cells Supporting Hepatitis C Virus RNA Replication

Journal of Virology ◽

10.1128/jvi.01565-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2182-2195 ◽

Cited By ~ 126

Author(s):

Paul Targett-Adams ◽

Steeve Boulant ◽

John McLauchlan

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Virus ◽

Three Dimensional ◽

Rna Replication ◽

Hcv Rna ◽

Double Stranded Rna ◽

Core Proteins ◽

Virus Rna ◽

Infected Cells

ABSTRACT The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.

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Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft

Journal of Virology ◽

10.1128/jvi.78.7.3480-3488.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3480-3488 ◽

Cited By ~ 247

Author(s):

Lu Gao ◽

Hideki Aizaki ◽

Jian-Wen He ◽

Michael M. C. Lai

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Raft ◽

Critical Role ◽

Rna Replication ◽

Dominant Negative ◽

Host Protein ◽

Hcv Rna ◽

Replication Complex ◽

Detergent Resistant Membranes

ABSTRACT The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.

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Binding of the La autoantigen to the hepatitis C virus 3′ untranslated region protects the RNA from rapid degradation in vitro

Journal of General Virology ◽

10.1099/0022-1317-82-1-113 ◽

2001 ◽

Vol 82 (1) ◽

pp. 113-120 ◽

Cited By ~ 52

Author(s):

Karin Spångberg ◽

Lisa Wiklund ◽

Stefan Schwartz

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Untranslated Region ◽

Negative Polarity ◽

Rna Degradation ◽

Hcv Rna ◽

Positive Polarity ◽

La Protein ◽

Infected Cells

We have analysed hepatitis C virus (HCV) RNAs in an in vitro RNA degradation assay. We found that the 3′ end of positive polarity HCV RNA is sensitive to cytosolic RNases whereas the 3′ end of negative polarity HCV RNA is relatively stable. Interaction of the HCV 3′ untranslated region with the cellular La protein prevented premature degradation of the HCV RNA. One may speculate that HCV RNAs interact with La protein in infected cells to prevent premature degradation of the viral RNAs.

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Knockdown of Autophagy Inhibits Infectious Hepatitis C Virus Release by the Exosomal Pathway

Journal of Virology ◽

10.1128/jvi.02383-15 ◽

2015 ◽

Vol 90 (3) ◽

pp. 1387-1396 ◽

Cited By ~ 75

Author(s):

Shubham Shrivastava ◽

Pradip Devhare ◽

Nanthiya Sujijantarat ◽

Robert Steele ◽

Young-Chan Kwon ◽

...

Keyword(s):

Immune Response ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Innate Immune Response ◽

Virus Transmission ◽

Infectious Hepatitis ◽

Innate Immune ◽

Hcv Rna ◽

Late Endosomes ◽

Infected Cells

ABSTRACTHepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. We showed previously that HCV induces autophagy for viral persistence by preventing the innate immune response. Knockdown of autophagy reduces extracellular HCV release, although the precise mechanism remains unknown. In this study, we observed that knockdown of autophagy genes enhances intracellular HCV RNA and accumulates infectious virus particles in cells. Since HCV release is linked with the exosomal pathway, we examined whether autophagy proteins associate with exosomes in HCV-infected cells. We observed an association between HCV and the exosomal marker CD63 in autophagy knockdown cells. Subsequently, we observed that levels of extracellular infectious HCV were significantly lower in exosomes released from autophagy knockdown cells. To understand the mechanism for reduced extracellular infectious HCV in the exosome, we observed that an interferon (IFN)-stimulated BST-2 gene is upregulated in autophagy knockdown cells and associated with the exosome marker CD63, which may inhibit HCV assembly or release. Taken together, our results suggest a novel mechanism involving autophagy and exosome-mediated HCV release from infected hepatocytes.IMPORTANCEAutophagy plays an important role in HCV pathogenesis. Autophagy suppresses the innate immune response and promotes survival of virus-infected hepatocytes. The present study examined the role of autophagy in secretion of infectious HCV from hepatocytes. Autophagy promoted HCV trafficking from late endosomes to lysosomes, thus providing a link with the exosome. Inhibition of HCV-induced autophagy could be used as a strategy to block exosome-mediated virus transmission.

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Polo-Like Kinase 1 Is Involved in Hepatitis C Virus Replication by Hyperphosphorylating NS5A

Journal of Virology ◽

10.1128/jvi.00068-10 ◽

2010 ◽

Vol 84 (16) ◽

pp. 7983-7993 ◽

Cited By ~ 58

Author(s):

Yung-Chia Chen ◽

Wen-Chi Su ◽

Jing-Ying Huang ◽

Ti-Chun Chao ◽

King-Song Jeng ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Kinase Inhibitor ◽

Rna Replication ◽

Cellular Growth ◽

Hcv Rna ◽

Reporter System ◽

Serine Threonine Kinase ◽

Infected Cells

ABSTRACT Hepatitis C virus (HCV) replication involves many viral and host factors. Here, we employed a lentivirus-based RNA interference (RNAi) screening approach to search for possible cellular factors. By using a kinase-phosphatase RNAi library and an HCV replicon reporter system, we identified a serine-threonine kinase, Polo-like kinase 1 (Plk1), as a potential host factor regulating HCV replication. Knockdown of Plk1 reduced both HCV RNA replication and nonstructural (NS) protein production in both HCV replicon cells and HCV-infected cells while it did not significantly affect host cellular growth or cell cycle. Overexpression of Plk1 in the knockdown cells rescued HCV replication. Interestingly, the ratio between the hyperphosphorylated form (p58) and the basal phosphorylated form (p56) of NS5A was lower in the Plk1 knockdown cells and Plk1 kinase inhibitor-treated cells than in the control groups. Further studies showed that Plk1 could be immunoprecipitated together with NS5A. Both proteins partially colocalized in the perinuclear region. Furthermore, Plk1 could phosphorylate NS5A to both the p58 and p56 forms in an in vitro assay system; the phosphorylation efficiency was comparable to that of the reported casein kinase. Taken together, this study shows that Plk1 is an NS5A phosphokinase and thereby indirectly regulates HCV RNA replication. Because of the differential effects of Plk1 on HCV replication and host cell growth, Plk1 could potentially serve as a target for anti-HCV therapy.

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Non-structural protein 4A of Hepatitis C virus accumulates on mitochondria and renders the cells prone to undergoing mitochondria-mediated apoptosis

Journal of General Virology ◽

10.1099/vir.0.81701-0 ◽

2006 ◽

Vol 87 (7) ◽

pp. 1935-1945 ◽

Cited By ~ 77

Author(s):

Yuki Nomura-Takigawa ◽

Motoko Nagano-Fujii ◽

Lin Deng ◽

Sohei Kitazawa ◽

Satoshi Ishido ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Hcv Rna ◽

Structural Protein ◽

Cellular Functions ◽

Replication Complex ◽

Huh7 Cells ◽

Infected Cells ◽

Rna Replicon

Non-structural protein 4A (NS4A) of Hepatitis C virus (HCV) functions as a cofactor for NS3 by forming a complex with it to augment its enzymic activities. NS4A also forms a complex with other HCV proteins, such as NS4B/NS5A, to facilitate the formation of the viral RNA replication complex on the endoplasmic reticulum (ER) membrane. In addition to its essential role in HCV replication, NS4A is thought to be involved in viral pathogenesis by affecting cellular functions. In this study, it was demonstrated that NS4A was localized not only on the ER, but also on mitochondria when expressed either alone or together with NS3 in the form of the NS3/4A polyprotein and in the context of HCV RNA replication in Huh7 cells harbouring an HCV RNA replicon. Moreover, NS4A expression altered the intracellular distribution of mitochondria significantly and caused mitochondrial damage, as evidenced by the collapsed mitochondrial transmembrane potential and release of cytochrome c into the cytoplasm, which led ultimately to induction of apoptosis through activation of caspase-3, but not caspase-8. Consistently, Huh7 cells expressing NS3/4A and those harbouring an HCV RNA replicon were shown to be more prone to undergoing actinomycin D-induced, mitochondria-mediated apoptosis, compared with the control Huh7 cells. Taken together, these results suggest the possibility that HCV exerts cytopathic effect (CPE) on the infected cells under certain conditions and that NS4A is responsible, at least in part, for the conditional CPE in HCV-infected cells.

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Modulation of Hepatitis C Virus RNA Abundance and the Isoprenoid Biosynthesis Pathway by MicroRNA miR-122 Involves Distinct Mechanisms

Journal of Virology ◽

10.1128/jvi.01156-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 666-670 ◽

Cited By ~ 80

Author(s):

Kara L. Norman ◽

Peter Sarnow

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mevalonate Pathway ◽

Direct Interaction ◽

Viral Rna ◽

Hcv Rna ◽

Isoprenoid Metabolism ◽

Virus Rna ◽

Intermediate Metabolites ◽

Rna Accumulation

ABSTRACT MicroRNA 122 (miR-122) promotes hepatitis C virus (HCV) RNA abundance through a direct interaction with the viral RNA and stimulates the mevalonate pathway in the animal liver. We found that overexpression of miR-122 enhanced viral RNA accumulation without affecting genes in the mevalonate pathway, such as the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) gene. However, inhibition of miR-122 decreased both HCV RNA and HMGCR RNA with little effects on the rates of HCV and HMGCR RNA synthesis. Loss of HCV RNA could not be restored by isoprenoid intermediate metabolites. Overall, these findings suggest that miR-122 modulates viral RNA abundance independently of its effect on isoprenoid metabolism.

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Novel Function of CD81 in Controlling Hepatitis C Virus Replication

Journal of Virology ◽

10.1128/jvi.02391-09 ◽

2010 ◽

Vol 84 (7) ◽

pp. 3396-3407 ◽

Cited By ~ 25

Author(s):

Yong-Yuan Zhang ◽

Bai-Hua Zhang ◽

Koji Ishii ◽

T. Jake Liang

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Entry ◽

Viral Protein ◽

Rna Replication ◽

Hcv Rna ◽

Viral Protein Synthesis ◽

Efficient Replication ◽

Infected Cells ◽

Kinetics Of

ABSTRACT The mechanisms of hepatitis C virus (HCV) replication remain poorly understood, and the cellular factors required for HCV replication are yet to be completely defined. CD81 is known to mediate HCV entry. Our study uncovered an unexpected novel function of CD81 in the HCV life cycle that is important for HCV RNA replication. HCV replication occurred efficiently in infected cells with high levels of CD81 expression. In HCV-infected or RNA-transfected cells with low levels of CD81 expression, initial viral protein synthesis occurred normally, but efficient replication failed to proceed. The aborted replication could be restored by the transient transfection of a CD81 expression plasmid. CD81-dependent replication was demonstrated with both an HCV infectious cell culture and HCV replicon cells of genotypes 1b and 2a. We also showed that CD81 expression is positively correlated with the kinetics of HCV RNA synthesis but inversely related to the kinetics of viral protein production, suggesting that CD81 may control viral replication by directing viral RNA template function to RNA replication. Thus, CD81 may be necessary for the efficient replication of the HCV genome in addition to its role in viral entry.

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Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus

Viruses ◽

10.3390/v11111039 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1039

Author(s):

Dandan Liu ◽

Philip R. Tedbury ◽

Shuiyun Lan ◽

Andrew D. Huber ◽

Maritza N. Puray-Chavez ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Viruses ◽

Drug Efficacy ◽

Confocal Imaging ◽

Hcv Rna ◽

Direct Acting Antivirals ◽

Infected Cells ◽

Positive Strand Rna ◽

Direct Acting

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((−)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (−)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (−)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action.

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