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

2004 ◽  
Vol 78 (7) ◽  
pp. 3480-3488 ◽  
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.

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 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 Genetic Interactions between Hepatitis C Virus Replicons

2004 ◽  
Vol 78 (21) ◽  
pp. 12085-12089 ◽  
Author(s):  
Matthew J. Evans ◽  
Charles M. Rice ◽  
Stephen P. Goff
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Replication ◽  
Genetic Interactions ◽  
Hcv Rna ◽  
Replication Complex ◽  
Huh7 Cells ◽  
High Level

ABSTRACT To investigate interactions between hepatitis C virus (HCV) RNA replication complexes, a system was developed to simultaneously select different HCV subgenomic replicons within the same cell. Transcomplementation of defective replicons was not observed, suggesting an isolated and independent nature of the HCV RNA replication complex. In contrast, a high level of competition between replicons was observed, such that the presence and increased fitness of one replicon reduced the capacity of a second one to stably replicate. These results suggest that at least one factor in Huh7 cells required for HCV RNA replication is limiting and saturable.

scholarly journals Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex

2009 ◽  
Vol 83 (13) ◽  
pp. 6554-6565 ◽  
Author(s):  
Zhe Liu ◽  
Feng Yang ◽  
Jason M. Robotham ◽  
Hengli Tang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Critical Role ◽  
Cyclophilin A ◽  
Hcv Rna ◽  
Nonstructural Proteins ◽  
Ppiase Activity ◽  
Replication Complex ◽  
And Function

ABSTRACT Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine's anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA's PPIase activity in the proper assembly and function of the HCV RC.

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

2006 ◽  
Vol 87 (7) ◽  
pp. 1935-1945 ◽  
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.

scholarly journals Interferon-α inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway

2001 ◽  
Vol 82 (4) ◽  
pp. 723-733 ◽  
Author(s):  
Michael Frese ◽  
Thomas Pietschmann ◽  
Darius Moradpour ◽  
Otto Haller ◽  
Ralf Bartenschlager
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Rna Replication ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Interferon Α ◽  
Hcv Rna ◽  
Dependent Manner

Hepatitis C virus (HCV) persists in the majority of infected individuals and is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Chronic hepatitis C is currently treated with interferon (IFN)-α or with a combination of IFN-α and ribavirin. The availability of an HCV replicon system (Lohmann et al., Science 285, 110–113, 1999) allowed the investigation of the effects of IFN on genuine HCV replication in cultured cells. It is shown here that IFN-α inhibits subgenomic HCV RNA replication in HuH-7 human hepatoma cells. Immunofluorescence, Western blot and Northern blot analysis revealed that levels of both HCV protein and replicon RNA were reduced after treatment with IFN-α in a dose-dependent manner. In further experiments, it was investigated whether MxA plays a role in the inhibition of HCV. The human MxA protein is an IFN-induced GTPase that has antiviral activity against various RNA viruses. However, HCV RNA replication was not affected in transfected HuH-7 cells that transiently overexpressed MxA. Moreover, a dominant-negative mutant of MxA did not interfere with the antiviral activity of IFN-α against HCV RNA replication. Taken together, these results demonstrate that IFN-α inhibits HCV replicons via an MxA-independent pathway.

scholarly journals Hepatitis C Virus RNA Replication Occurs on a Detergent-Resistant Membrane That Cofractionates with Caveolin-2

2003 ◽  
Vol 77 (7) ◽  
pp. 4160-4168 ◽  
Author(s):  
Stephanie T. Shi ◽  
Ki-Jeong Lee ◽  
Hideki Aizaki ◽  
Soon B. Hwang ◽  
Michael M. C. Lai
Keyword(s):  
Protein Synthesis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Raft ◽  
Rna Synthesis ◽  
De Novo ◽  
Rna Replication ◽  
Viral Rna ◽  
Hcv Rna ◽  
Huh7 Cells

ABSTRACT The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly understood. In this study, we labeled de novo-synthesized viral RNA in situ with bromouridine triphosphate (BrUTP) in Huh7 cells expressing an HCV subgenomic replicon. By immunofluorescence staining using an anti-BrUTP antibody and confocal microscopy, we showed that the newly synthesized HCV RNA was localized to distinct speckle-like structures, which also contain all of the HCV nonstructural (NS) proteins. These speckles are distinct from lipid droplets and are separated from the endoplasmic reticulum (ER), where some HCV NS proteins also reside. Membrane flotation analysis demonstrated that almost all of the NS5A and part of the NS5B proteins and all of the viral RNA were present in membrane fractions which are resistant to treatment with 1% NP-40 at 4°C. They were cofractionated with caveolin-2, a lipid-raft-associated intracellular membrane protein, in the presence or absence of the detergent. In contrast, the ER-resident proteins were detergent soluble. These properties suggest that the membranes on which HCV RNA replication occurs are lipid rafts recruited from the intracellular membranes. The protein synthesis inhibitors cycloheximide and puromycin did not inhibit viral RNA synthesis, indicating that HCV RNA replication does not require continuous protein synthesis. We suggest that HCV RNA synthesis occurs on a lipid raft membrane structure.

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

2019 ◽  
Author(s):  
Camille Baudesson ◽  
Céline Amadori ◽  
Hassan Danso ◽  
Flora Donati ◽  
Quentin Nevers ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Direct Interaction ◽  
Micro Rna ◽  
Host Protein ◽  
Hcv Rna ◽  
Serine Residue ◽  
Reciprocal Regulation ◽  
Mrna Targets ◽  
Infected Cells

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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