scholarly journals Hepatitis C Virus (HCV) Induces Formation of Stress Granules Whose Proteins Regulate HCV RNA Replication and Virus Assembly and Egress

2012 ◽  
Vol 86 (20) ◽  
pp. 11043-11056 ◽  
Author(s):  
U. Garaigorta ◽  
M. H. Heim ◽  
B. Boyd ◽  
S. Wieland ◽  
F. V. Chisari
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Assembly ◽  
Stress Granules ◽  
Rna Replication ◽  
Hcv Rna

scholarly journals Palmitoylation of Hepatitis C Virus NS2 Regulates Its Subcellular Localization and NS2-NS3 Autocleavage

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
Ming-Jhan Wu ◽  
Saravanabalaji Shanmugam ◽  
Christoph Welsch ◽  
MinKyung Yi
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Envelope Protein ◽  
Virus Assembly ◽  
Rna Replication ◽  
Encephalomyocarditis Virus ◽  
Hcv Rna ◽  
Particle Assembly ◽  
Infectious Particle ◽  
Multiple Functions

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly. IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.

scholarly journals Regulating Intracellular Antiviral Defense and Permissiveness to Hepatitis C Virus RNA Replication through a Cellular RNA Helicase, RIG-I

2005 ◽  
Vol 79 (5) ◽  
pp. 2689-2699 ◽  
Author(s):  
Rhea Sumpter ◽  
Yueh-Ming Loo ◽  
Eileen Foy ◽  
Kui Li ◽  
Mitsutoshi Yoneyama ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Infections ◽  
Cultured Cells ◽  
Rna Replication ◽  
Rna Helicase ◽  
Hcv Rna ◽  
Helicase Domain ◽  
Replication Studies ◽  
Virus Rna

ABSTRACT Virus-responsive signaling pathways that induce alpha/beta interferon production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates interferon regulatory factor 3 (IRF3), thereby inducing interferon in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an interferon-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF3 by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an interferon-responsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.

scholarly journals An N-Terminal Amphipathic Helix in Hepatitis C Virus (HCV) NS4B Mediates Membrane Association, Correct Localization of Replication Complex Proteins, and HCV RNA Replication

2004 ◽  
Vol 78 (20) ◽  
pp. 11393-11400 ◽  
Author(s):  
Menashe Elazar ◽  
Ping Liu ◽  
Charles M. Rice ◽  
Jeffrey S. Glenn
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Replication ◽  
Hcv Rna ◽  
Membrane Association ◽  
Amphipathic Helix ◽  
Nonstructural Proteins ◽  
Replication Complex ◽  
Cytoplasmic Membranes ◽  
Positive Strand Rna

ABSTRACT Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the virus's nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AH's ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4B's N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies.

scholarly journals Novel Robust Hepatitis C Virus Mouse Efficacy Model

2006 ◽  
Vol 50 (10) ◽  
pp. 3260-3268 ◽  
Author(s):  
Qing Zhu ◽  
Yoko Oei ◽  
Dirk B. Mendel ◽  
Evelyn N. Garrett ◽  
Montesa B. Patawaran ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protease Inhibitor ◽  
Small Animal ◽  
Rna Replication ◽  
Treatment Withdrawal ◽  
Whole Body ◽  
Hcv Rna ◽  
Whole Body Imaging

ABSTRACT The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line (T7-11). The model was validated by demonstrating that both a small-molecule NS3/4A protease inhibitor (BILN 2061) and human alpha interferon (IFN-α) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. We further showed that protease inhibitor and IFN-α combination therapy was more effective in reducing HCV RNA replication than treatment with each compound alone and supports testing in humans. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo as part of aggressive drug discovery efforts.

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

2007 ◽  
Vol 82 (5) ◽  
pp. 2182-2195 ◽  
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.

scholarly journals Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines

2012 ◽  
Vol 93 (7) ◽  
pp. 1422-1431 ◽  
Author(s):  
Midori Takeda ◽  
Masanori Ikeda ◽  
Yasuo Ariumi ◽  
Takaji Wakita ◽  
Nobuyuki Kato
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Rna Replication ◽  
Hcv Rna ◽  
Reporter Assay ◽  
Hepatoma Cell Lines

A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann–Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.

scholarly journals Palmitoylation and Polymerization of Hepatitis C Virus NS4B Protein

2006 ◽  
Vol 80 (12) ◽  
pp. 6013-6023 ◽  
Author(s):  
Guann-Yi Yu ◽  
Ki-Jeong Lee ◽  
Lu Gao ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Membrane Structure ◽  
Rna Replication ◽  
Polymerization Process ◽  
Cysteine Residues ◽  
Hcv Rna ◽  
Subgenomic Replicon ◽  
Lipid Modifications ◽  
Ns4b Protein

ABSTRACT Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.

scholarly journals Efficient Rescue of Hepatitis C Virus RNA Replication by trans-Complementation with Nonstructural Protein 5A

2005 ◽  
Vol 79 (2) ◽  
pp. 896-909 ◽  
Author(s):  
Nicole Appel ◽  
Ulrike Herian ◽  
Ralf Bartenschlager
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Functional Organization ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Hcv Rna ◽  
Replication Complex ◽  
Diarrhea Virus ◽  
Amino Terminal ◽  
Essential Components

ABSTRACT Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.

scholarly journals Arsenic Trioxide Inhibits Hepatitis C Virus RNA Replication through Modulation of the Glutathione Redox System and Oxidative Stress

2008 ◽  
Vol 83 (5) ◽  
pp. 2338-2348 ◽  
Author(s):  
Misao Kuroki ◽  
Yasuo Ariumi ◽  
Masanori Ikeda ◽  
Hiromichi Dansako ◽  
Takaji Wakita ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Arsenic Trioxide ◽  
Redox System ◽  
Rna Replication ◽  
Hcv Rna ◽  
Glutathione Redox System ◽  
And Oxidative Stress ◽  
Glutathione Redox

ABSTRACT Arsenic trioxide (ATO), a therapeutic reagent used for the treatment of acute promyelocytic leukemia, has recently been reported to increase human immunodeficiency virus type 1 infectivity. However, in this study, we have demonstrated that replication of genome-length hepatitis C virus (HCV) RNA (O strain of genotype 1b) was notably inhibited by ATO at submicromolar concentrations without cell toxicity. RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were also inhibited by ATO after the HCV infection. To clarify the mechanism of the anti-HCV activity of ATO, we examined whether or not PML is associated with this anti-HCV activity, since PML is known to be a target of ATO. Interestingly, we observed the cytoplasmic translocation of PML after treatment with ATO. However, ATO still inhibited the HCV RNA replication even in the PML knockdown cells, suggesting that PML is dispensable for the anti-HCV activity of ATO. In contrast, we found that N-acetyl-cysteine, an antioxidant and glutathione precursor, completely and partially eliminated the anti-HCV activity of ATO after 24 h and 72 h of treatment, respectively. In this context, it is worth noting that we found an elevation of intracellular superoxide anion radical, but not hydrogen peroxide, and the depletion of intracellular glutathione in the ATO-treated cells. Taken together, these findings suggest that ATO inhibits the HCV RNA replication through modulation of the glutathione redox system and oxidative stress.

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