scholarly journals Cellular RNA Helicase p68 Relocalization and Interaction with the Hepatitis C Virus (HCV) NS5B Protein and the Potential Role of p68 in HCV RNA Replication

2004 ◽  
Vol 78 (10) ◽  
pp. 5288-5298 ◽  
Author(s):  
Phuay-Yee Goh ◽  
Yee-Joo Tan ◽  
Siew Pheng Lim ◽  
Y. H. Tan ◽  
Seng Gee Lim ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Line ◽  
Rna Helicase ◽  
Hcv Rna ◽  
Activity Levels ◽  
Nonstructural Proteins ◽  
Negative Strand ◽  
Replicon Cell Line ◽  
Rna Helicase P68

ABSTRACT Chronic infection by hepatitis C virus (HCV) can lead to severe hepatitis and cirrhosis and is closely associated with hepatocellular carcinoma. The replication cycle of HCV is poorly understood but is likely to involve interaction with host factors. In this report, we show that NS5B, the HCV RNA-dependent RNA polymerase (RdRp), interacts with a human RNA helicase, p68. Transient expression of NS5B alone, as well as the stable expression of all the nonstructural proteins in a HCV replicon-bearing cell line (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110-113), causes the redistribution of endogenous p68 from the nucleus to the cytoplasm. Deletion of the C-terminal two-thirds of NS5B (NS5BΔC) dramatically reduces its coimmunoprecipitation (co-IP) with endogenous p68, while the deletion of the N-terminal region (NS5BΔN1 and NS5BΔN2) does not affect its interaction with p68. In consistency with the co-IP results, NS5BΔC does not cause the relocalization of p68 whereas NS5BΔN1 does. With a replicon cell line, we were not able to detect a change in positive- and negative-strand synthesis when p68 levels were reduced using small interfering RNA (siRNA). In cells transiently transfected with a full-length HCV construct, however, the depletion (using specific p68 siRNA) of endogenous p68 correlated with a reduction in the transcription of negative-strand from positive-strand HCV RNA. Overexpression of NS5B and NS5BΔN1, but not that of NS5BΔC, causes a reduction in the negative-strand synthesis, indicating that overexpressed NS5B and NS5BΔN1 sequesters p68 from the replication complexes (thus reducing their replication activity levels). Identification of p68 as a cellular factor involved in HCV replication, at least for cells transiently transfected with a HCV expression construct, is a step towards understanding HCV replication.

scholarly journals A Cell-Based Model of HCV-Negative-Strand RNA Replication Utilizing a Chimeric Hepatitis C Virus/Reporter RNA Template

2002 ◽  
Vol 13 (6) ◽  
pp. 353-362 ◽  
Author(s):  
Robert W King ◽  
Marianne Zecher ◽  
Matthew W Jeffries ◽  
Denise R Carroll ◽  
Joseph M Parisi ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Cell Line ◽  
Rna Replication ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
Negative Strand ◽  
A Cell ◽  
Chimeric Rna

The inability of hepatitis C virus (HCV) to replicate in cell culture has hindered the discovery of antiviral agents against this virus. One of the biggest challenges has been to find a model that allows one to easily and accurately quantify the level of HCV RNA replication that is occurring inside the cell. In an attempt to solve this problem, we have created a plasmid pMJ050 that encodes a chimeric ‘HCV-like’ RNA that can act as a reporter for HCV RNA replication. This RNA consists of an antisense copy of the firefly luciferase sequence flanked by the 5′ and 3′ untranslated regions of the negative strand of the HCV RNA. If, in cells that contain functional HCV proteins, the chimeric RNA is recognized as a substrate for the viral RNA-dependent RNA polymerase, the chimeric RNA will be transcribed into the complementary strand. This RNA has a 5′ HCV internal ribosome entry site and the luciferase sequence in the coding orientation, allowing translation of the RNA into biologically active luciferase. When pMJ050 was transfected into a cell line that is stably transfected with a cDNA copy of the HCV 1b genome, luciferase was produced in a manner that was dependent upon the presence of at least a functional HCV RNA-dependent RNA polymerase. In addition, we constructed a cell line, 293B4α that constitutively produced luciferase in response to the presence of functional HCV proteins. This system permits the accurate determination of the level of HCV RNA replication by the quantification of luciferase.

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 Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3′-Terminal Sequences

2002 ◽  
Vol 76 (19) ◽  
pp. 9563-9574 ◽  
Author(s):  
Robert M. Smith ◽  
Cherie M. Walton ◽  
Catherine H. Wu ◽  
George Y. Wu
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Secondary Structure ◽  
Rna Structure ◽  
Genome Stability ◽  
Rnase H ◽  
Hcv Rna ◽  
Positive Strand ◽  
Negative Strand

ABSTRACT The 3′-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3′ untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3′ terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3′-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3′-X region RNA in vitro.

scholarly journals Ribavirin Resistance in Hepatitis C Virus Replicon-Containing Cell Lines Conferred by Changes in the Cell Line or Mutations in the Replicon RNA

2005 ◽  
Vol 79 (4) ◽  
pp. 2346-2355 ◽  
Author(s):  
Julie K. Pfeiffer ◽  
Karla Kirkegaard
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Types ◽  
Resistant Cell ◽  
Resistant Isolate ◽  
Hcv Rna ◽  
Replicon Cell ◽  
Error Accumulation

ABSTRACT Ribavirin (RBV), used in combination with alpha interferon to treat hepatitis C virus (HCV) infections, is a guanosine nucleotide analog that can increase the error rate of viral RNA-dependent RNA polymerases, imbalance intracellular nucleotide pools, and cause toxicity in many cell types. To determine potential mechanisms of RBV resistance during HCV RNA replication, we passaged HCV replicon-containing cell lines in the presence of increasing concentrations of RBV. RBV-resistant, HCV replicon-containing cell lines were generated, and the majority of RBV resistance was found to be conferred by changes in the cell lines. The resistant cell lines were defective in RBV import, as measured by [3H]RBV uptake experiments. These cell lines displayed reduced RBV toxicity and reduced error accumulation during infection with poliovirus, whose replication is known to be sensitive to RBV-induced error. For one RBV-resistant isolate, two mutations in the replicon RNA contributed to the observed phenotype. Two responsible mutations resided in the C-terminal region of NS5A, G404S, and E442G and were each sufficient for low-level RBV resistance. Therefore, RBV resistance in HCV replicon cell lines can be conferred by changes in the cell line or by mutations in the HCV replicon.

scholarly journals Hepatitis C Virus Persistence after Spontaneous or Treatment-Induced Resolution of Hepatitis C

2004 ◽  
Vol 78 (11) ◽  
pp. 5867-5874 ◽  
Author(s):  
Tram N. Q. Pham ◽  
Sonya A. MacParland ◽  
Patricia M. Mulrooney ◽  
Helen Cooksley ◽  
Nikolai V. Naoumov ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mononuclear Cells ◽  
Lymphoid Cells ◽  
Nucleic Acid Hybridization ◽  
Hcv Rna ◽  
Rt Pcr ◽  
Virus Eradication ◽  
Peripheral Blood Mononuclear ◽  
Negative Strand

ABSTRACT It is presumed that resolution of hepatitis C, as evidenced by normalization of liver function tests and disappearance of hepatitis C virus (HCV) RNA from serum, as determined by conventional laboratory assays, reflects virus eradication. In this study, we examined the expression of the HCV genome in the sera, peripheral blood mononuclear cells (PBMC), and, on some occasions, monocyte-derived dendritic cells (DC) long after resolution of hepatitis C by using a highly sensitive reverse transcription (RT)-PCR-nucleic acid hybridization (RT-PCR-NAH) assay. The samples obtained from 16 randomly selected patients (5 with spontaneous and 11 with treatment-induced resolution), monitored for up to 5 years, were studied by qualitative and semiquantitative RT-PCR-NAH and by real-time RT-PCR to detect the HCV RNA positive strand. The replicative HCV RNA negative strand was examined in PBMC after culture with a T-cell proliferation stimulating mitogen. The findings show that HCV RNA was carried in the convalescent-phase sera and/or PBMC in all 16 individuals investigated. Also, DC from six of seven patients were reactive for the HCV genome. Importantly, traces of the HCV RNA negative strand, suggesting progressing virus replication, were detected in the majority of mitogen-stimulated PBMC, including four samples collected 5 years after recovery. Sequencing of the HCV 5′ untranslated region fragment revealed genotype 1b in four of nine individuals examined and genotypes 1a and 2a in three and two patients, respectively. These results imply that HCV RNA can persist at very low levels in the serum and peripheral lymphoid cells and that an intermediate replicative form of the HCV genome can persist in PBMC for many years after apparently complete spontaneous or antiviral therapy-induced resolution of chronic hepatitis C.

scholarly journals Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA

2003 ◽  
Vol 84 (10) ◽  
pp. 2761-2769 ◽  
Author(s):  
Nazira El-Hage ◽  
Guangxiang Luo
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hepatoma Cell ◽  
Viral Proteins ◽  
Subcellular Fractionation ◽  
Hcv Rna ◽  
Hepatoma Cell Line ◽  
Nonstructural Proteins ◽  
Replication Complex ◽  
Membrane Bound

Biochemical studies revealed that nonstructural proteins of hepatitis C virus (HCV) interacted with each other and were associated with intracellular membranes. The goals of this study were to determine whether nonstructural viral proteins are colocalized at specific intracellular sites where HCV RNA is replicated and to identify the virus components of the HCV replication complex (RC). Immunofluorescence and subcellular fractionation studies were performed to determine the intracellular colocalization of nonstructural HCV proteins and the replicating RNA in a human hepatoma cell line, Huh7, in which a subgenomic HCV RNA was replicated persistently. The replicating HCV RNA was labelled with 5-bromouridine 5′-triphosphate (BrUTP). Results show that each of the nonstructural HCV proteins was colocalized predominantly with the newly synthesized HCV RNA labelled with BrUTP and an endoplasmic reticulum (ER) protein, calnexin. Consistent with these findings, subcellular fractionation and Western blot analyses revealed that the nonstructural HCV proteins were colocalized with HCV RNA mainly in the membrane fractions. Conversely, the viral nonstructural proteins and RNA remained in the soluble fractions upon treatment with detergent, confirming the membrane association of the HCV RC. HCV RNA in the membrane-bound RC was resistant to RNase treatment, whereas it became sensitive to RNases once the membranes were disrupted by treatment with detergent, suggesting that the HCV RC is assembled within membrane structures. Collectively, these findings demonstrate that HCV RNA replication occurs in the perinuclear ER membrane-bound HCV RC, containing nonstructural viral proteins and RNA.

scholarly journals In Vitro RNA Replication Directed by Replicase Complexes Isolated from the Subgenomic Replicon Cells of Hepatitis C Virus

2003 ◽  
Vol 77 (3) ◽  
pp. 2295-2300 ◽  
Author(s):  
Vicky C. H. Lai ◽  
Shannon Dempsey ◽  
Johnson Y. N. Lau ◽  
Zhi Hong ◽  
Weidong Zhong
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
Divalent Cations ◽  
Hcv Rna ◽  
Nonstructural Proteins ◽  
Subgenomic Replicon ◽  
Free System ◽  
Potential Inhibitors

ABSTRACT Replication of hepatitis C virus (HCV) RNA in virus-infected cells is believed to be catalyzed by viral replicase complexes (RCs), which may consist of various virally encoded nonstructural proteins and host factors. In this study, we characterized the RC activity of a crude membrane fraction isolated from HCV subgenomic replicon cells. The RC preparation was able to use endogenous replicon RNA as a template to synthesize both single-stranded (ss) and double-stranded (ds) RNA products. Divalent cations (Mg2+ and Mn2+) showed different effects on RNA synthesis. Mg2+ ions stimulated the synthesis of ss RNA but had little effect on the synthesis of ds RNA. In contrast, Mn2+ ions enhanced primarily the synthesis of ds RNA. Interestingly, ss RNA could be synthesized under certain conditions in the absence of ds RNA, and vice versa, suggesting that the ss and ds RNA were derived either from different forms of replicative intermediates or from different RCs. Pulse-chase analysis showed that radioactivity incorporated into the ss RNA was chased into the ds RNA and other larger RNA species. This observation indicated that the newly synthesized ss RNA could serve as a template for a further round of RNA synthesis. Finally, 3′ deoxyribonucleoside triphosphates were able to inhibit RNA synthesis in this cell-free system, presumably through chain termination, with 3′ dGTP having the highest potency. Establishment of the replicase assay will facilitate the identification and evaluation of potential inhibitors that would act against the entire RC of HCV.

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.

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