Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5' Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome

ABSTRACTHigh-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein involved in multiple human diseases, including infectious diseases, immune disorders, metabolic disorders, and cancer. HMGB1 is comprised of two tandem HMG boxes (the A box and the B box) containing DNA-binding domains and an acidic C-terminal peptide. It has been reported that HMGB1 enhances viral replication by binding to viral proteins. However, its role in hepatitis C virus (HCV) replication is unknown. Here, we show that HMGB1 promoted HCV replication but had no effect on HCV translation. RNA immunoprecipitation experiments indicated that the positive strand, not the negative strand, of HCV RNA interacted with HMGB1. HCV infection triggered HMGB1 protein translocation from the nucleus to the cytoplasm, in which it interacted with the HCV genome. Moreover, the A box of HMGB1 is the pivotal domain to interact with stem-loop 4 (SL4) of the HCV 5′ untranslated region. Deletion of the HMGB1 A box abrogated the enhancement of HCV replication by HMGB1. Our data suggested that HMGB1 serves as a proviral factor of HCV to facilitate viral replication in hepatocytes by interaction with the HCV genome.IMPORTANCEHepatitis C virus (HCV) is a major global health threat, affecting more than 170 million people infection worldwide. These patients are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Currently, no vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. In this study, we found a novel HCV RNA-binding protein, HMGB1, that promotes HCV RNA replication. Moreover, SL4 in the 5′ untranslated region of the HCV genome is the key region for HMGB1 binding, and the A box of HMGB1 protein is the functional domain to interact with HCV RNA and enhance viral replication. HMGB1 appears to play an important role in HCV-related diseases, and further investigation is warranted to elucidate the specific actions of HMGB1 in HCV pathogenesis.

Download Full-text

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

Journal of Virology ◽

10.1128/jvi.01714-15 ◽

2015 ◽

Vol 89 (22) ◽

pp. 11356-11371 ◽

Cited By ~ 32

Author(s):

Shivaprasad Shwetha ◽

Anuj Kumar ◽

Ranajoy Mullick ◽

Deeptha Vasudevan ◽

Nilanjan Mukherjee ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Replication ◽

Untranslated Region ◽

Rna Synthesis ◽

Binding Protein ◽

Viral Rna ◽

Host Protein ◽

Polypyrimidine Tract ◽

Polypyrimidine Tract Binding Protein

ABSTRACTHuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3′ untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3′ UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3′ UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3′ UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR.IMPORTANCEHepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3′ untranslated region (UTR) of HCV RNA. At the 3′ UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

Download Full-text

Faculty Opinions recommendation of Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725354595.793504417 ◽

2015 ◽

Author(s):

Patricia C Weber

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Replication ◽

Rna Replication ◽

Structural Basis

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.79.5.2689-2699.2005 ◽

2005 ◽

Vol 79 (5) ◽

pp. 2689-2699 ◽

Cited By ~ 641

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.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.78.20.11393-11400.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 11393-11400 ◽

Cited By ~ 112

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.

Download Full-text

Novel Robust Hepatitis C Virus Mouse Efficacy Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00413-06 ◽

2006 ◽

Vol 50 (10) ◽

pp. 3260-3268 ◽

Cited By ~ 22

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.

Download Full-text

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.

Download Full-text

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

Journal of General Virology ◽

10.1099/vir.0.040725-0 ◽

2012 ◽

Vol 93 (7) ◽

pp. 1422-1431 ◽

Cited By ~ 8

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.

Download Full-text

Palmitoylation and Polymerization of Hepatitis C Virus NS4B Protein

Journal of Virology ◽

10.1128/jvi.00053-06 ◽

2006 ◽

Vol 80 (12) ◽

pp. 6013-6023 ◽

Cited By ~ 86

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.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.79.2.896-909.2005 ◽

2005 ◽

Vol 79 (2) ◽

pp. 896-909 ◽

Cited By ~ 71

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.

Download Full-text