scholarly journals Role of the 5′-Proximal Stem-Loop Structure of the 5′ Untranslated Region in Replication and Translation of Hepatitis C Virus RNA

2003 ◽  
Vol 77 (5) ◽  
pp. 3312-3318 ◽  
Author(s):  
Guangxiang Luo ◽  
Shaojie Xin ◽  
Zhaohui Cai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Replication ◽  
Hcv Rna ◽  
Loop Structure ◽  
Nucleotide Substitutions ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Compensatory Mutations ◽  
Cell Colony

ABSTRACT Sequences of the untranslated regions at the 5′ and 3′ ends (5′UTR and 3′UTR) of the hepatitis C virus (HCV) RNA genome are highly conserved and contain cis-acting RNA elements for HCV RNA replication. The HCV 5′UTR consists of two distinct RNA elements, a short 5′-proximal stem-loop RNA element (nucleotides 1 to 43) and a longer element of internal ribosome entry site. To determine the sequence and structural requirements of the 5′-proximal stem-loop RNA element in HCV RNA replication and translation, a mutagenesis analysis was preformed by nucleotide deletions and substitutions. Effects of mutations in the 5′-proximal stem-loop RNA element on HCV RNA replication were determined by using a cell-based HCV replicon replication system. Deletion of the first 20 nucleotides from the 5′ end resulted in elimination of cell colony formation. Likewise, disruption of the 5′-proximal stem-loop by nucleotide substitutions abolished the ability of HCV RNA to induce cell colony formation. However, restoration of the 5′-proximal stem-loop by compensatory mutations with different nucleotides rescued the ability of the subgenomic HCV RNA to replicate in Huh7 cells. In addition, deletion and nucleotide substitutions of the 5′-proximal stem-loop structure, including the restored stem-loop by compensatory mutations, all resulted in reduction of translation by two- to fivefold, suggesting that the 5′-proximal stem-loop RNA element also modulates HCV RNA translation. These findings demonstrate that the 5′-proximal stem-loop of the HCV RNA is a cis-acting RNA element that regulates HCV RNA replication and translation.

scholarly journals Specific Interaction of Hepatitis C Virus Protease/Helicase NS3 with the 3′-Terminal Sequences of Viral Positive- and Negative-Strand RNA

2001 ◽  
Vol 75 (4) ◽  
pp. 1708-1721 ◽  
Author(s):  
Rajeev Banerjee ◽  
Asim Dasgupta
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Specific Interaction ◽  
Helicase Domain ◽  
Loop Structure ◽  
Stem Loop ◽  
Negative Strand ◽  
Stem Loop Structure ◽  
Rna Interaction ◽  
Positive Strand Rna

ABSTRACT The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and ΔpNS3 (helicase domain only) and examined their abilities to interact with the 3′-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3′ untranslated region (UTR). The results presented here demonstrate that NS3 (and ΔpNS3) interacts efficiently and specifically with the 3′-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5′-terminal RNA sequences. The interaction of NS3 with the 3′-terminal negative strand [called 3′(−) UTR127] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3′ terminus (nucleotides 5 to 20 from the 3′ end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and ΔpNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3′-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3′(−) UTR binding, specific interaction of NS3 (or ΔpNS3) with the 3′-terminal sequences of the positive-strand RNA [3′(+) UTR] appears to require the entire 3′(+) UTR of HCV. Deletion of either the 98-nucleotide 3′-terminal conserved region or the 5′ half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3′-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.

scholarly journals Effects of Mutations of the Initiation Nucleotides on Hepatitis C Virus RNA Replication in the Cell

2004 ◽  
Vol 78 (7) ◽  
pp. 3633-3643 ◽  
Author(s):  
Zhaohui Cai ◽  
T. Jake Liang ◽  
Guangxiang Luo
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Colony Formation ◽  
Rna Viruses ◽  
Rna Replication ◽  
Hcv Rna ◽  
Positive Strand ◽  
Negative Strand ◽  
Huh7 Cells ◽  
Cell Colony

ABSTRACT Replication of nearly all RNA viruses depends on a virus-encoded RNA-dependent RNA polymerase (RdRp). Our earlier work found that purified recombinant hepatitis C virus (HCV) RdRp (NS5B) was able to initiate RNA synthesis de novo by using purine (A and G) but not pyrimidine (C and U) nucleotides (G. Luo et al., J. Virol. 74:851-863, 2000). For most human RNA viruses, the initiation nucleotides of both positive- and negative-strand RNAs were found to be either an adenylate (A) or guanylate (G). To determine the nucleotide used for initiation and control of HCV RNA replication, a genetic mutagenesis analysis of the nucleotides at the very 5′ and 3′ ends of HCV RNAs was performed by using a cell-based HCV replicon replication system. Either a G or an A at the 5′ end of HCV genomic RNA was able to efficiently induce cell colony formation, whereas a nucleotide C at the 5′ end dramatically reduced the efficiency of cell colony formation. Likewise, the 3′-end nucleotide U-to-C mutation did not significantly affect the efficiency of cell colony formation. In contrast, a U-to-G mutation at the 3′ end caused a remarkable decrease in cell colony formation, and a U-to-A mutation resulted in a complete abolition of cell colony formation. Sequence analysis of the HCV replicon RNAs recovered from G418-resistant Huh7 cells revealed several interesting findings. First, the 5′-end nucleotide G of the replicon RNA was changed to an A upon multiple rounds of replication. Second, the nucleotide A at the 5′ end was stably maintained among all replicon RNAs isolated from Huh7 cells transfected with an RNA with a 5′-end A. Third, initiation of HCV RNA replication with a CTP resulted in a >10-fold reduction in the levels of HCV RNAs, suggesting that initiation of RNA replication with CTP was very inefficient. Fourth, the 3′-end nucleotide U-to-C and -G mutations were all reverted back to a wild-type nucleotide U. In addition, extra U and UU residues were identified at the 3′ ends of revertants recovered from Huh7 cells transfected with an RNA with a nucleotide G at the 3′ end. We also determined the 5′-end nucleotide of positive-strand RNA of some clinical HCV isolates. Either G or A was identified at the 5′ end of HCV RNA genome depending on the specific HCV isolate. Collectively, these findings demonstrate that replication of positive-strand HCV RNA was preferentially initiated with purine nucleotides (ATP and GTP), whereas the negative-strand HCV RNA replication is invariably initiated with an ATP.

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 Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12008-12022 ◽  
Author(s):  
Brandon L. Walter ◽  
Todd B. Parsley ◽  
Ellie Ehrenfeld ◽  
Bert L. Semler
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Protein ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

ABSTRACT The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

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 HMGB1 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 4 in the Viral 5′ Untranslated Region

2015 ◽  
Vol 90 (5) ◽  
pp. 2332-2344 ◽  
Author(s):  
Rong Yu ◽  
Darong Yang ◽  
Shaohua Lei ◽  
Xiaohong Wang ◽  
Xianghe Meng ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Untranslated Region ◽  
Rna Binding ◽  
High Mobility ◽  
Hcv Rna ◽  
Functional Domain ◽  
Hmgb1 Protein ◽  
Stem Loop

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.

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