scholarly journals Alternative Approaches for Efficient Inhibition of Hepatitis C Virus RNA Replication by Small Interfering RNAs

2004 ◽  
Vol 78 (7) ◽  
pp. 3436-3446 ◽  
Author(s):  
Jan Krönke ◽  
Ralf Kittler ◽  
Frank Buchholz ◽  
Marc P. Windisch ◽  
Thomas Pietschmann ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Rna Replication ◽  
Similar Degree ◽  
Hcv Rna ◽  
Target Sequence ◽  
Small Interfering Rnas ◽  
Conserved Sequence ◽  
Nontranslated Region

ABSTRACT Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It has recently been shown that HCV RNA replication is susceptible to small interfering RNAs (siRNAs), but the antiviral activity of siRNAs depends very much on their complementarity to the target sequence. Thus, the high degree of sequence diversity between different HCV genotypes and the rapid evolution of new quasispecies is a major problem in the development of siRNA-based gene therapies. For this study, we developed two alternative strategies to overcome these obstacles. In one approach, we used endoribonuclease-prepared siRNAs (esiRNAs) to simultaneously target multiple sites of the viral genome. We show that esiRNAs directed against various regions of the HCV coding sequence as well as the 5′ nontranslated region (5′ NTR) efficiently block the replication of subgenomic and genomic HCV replicons. In an alternative approach, we generated pseudotyped retroviruses encoding short hairpin RNAs (shRNAs). A total of 12 shRNAs, most of them targeting highly conserved sequence motifs within the 5′ NTR or the early core coding region, were analyzed for their antiviral activities. After the transduction of Huh-7 cells containing a subgenomic HCV replicon, we found that all shRNAs targeting sequences in domain IV or nearby coding sequences blocked viral replication. In contrast, only one of seven shRNAs targeting sequences in domain II or III had a similar degree of antiviral activity, indicating that large sections of the NTRs are resistant to RNA interference. Moreover, we show that naive Huh-7 cells that stably expressed certain 5′ NTR-specific shRNAs were largely resistant to a challenge with HCV replicons. These results demonstrate that the retroviral transduction of HCV-specific shRNAs provides a new possibility for antiviral intervention.

scholarly journals Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

2001 ◽  
Vol 75 (24) ◽  
pp. 12047-12057 ◽  
Author(s):  
Peter Friebe ◽  
Volker Lohmann ◽  
Nicole Krieger ◽  
Ralf Bartenschlager
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Virus ◽  
Rna Replication ◽  
Hcv Rna ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Nontranslated Region ◽  
High Level ◽  
Hcv Ires

ABSTRACT Sequences in the 5′ and 3′ termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5′ end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5′ NTR required for RNA replication. We show that deletions introduced into the 5′ terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5′ NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5′ NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5′ NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5′ NTR of HCV RNA essential for replication of this virus.

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 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 Short-Term Monotherapy with IDX184, a Liver-Targeted Nucleotide Polymerase Inhibitor, in Patients with Chronic Hepatitis C Virus Infection

2012 ◽  
Vol 56 (12) ◽  
pp. 6372-6378 ◽  
Author(s):  
Jacob Lalezari ◽  
David Asmuth ◽  
Arnaldo Casiró ◽  
Hugo Vargas ◽  
Shannon Lawrence ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Viral Load ◽  
Hepatitis C ◽  
Adverse Events ◽  
Antiviral Activity ◽  
Chronic Hepatitis C ◽  
Hcv Rna ◽  
Plasma Exposure ◽  
Chronic Hepatitis C Virus

ABSTRACTIDX184 is a liver-targeted prodrug of 2′-methylguanosine (2′-MeG) monophosphate. This study investigated the safety, tolerability, antiviral activity, and pharmacokinetics of IDX184 as a single agent in treatment-naïve patients with genotype-1 chronic hepatitis C virus (HCV) infection. Forty-one patients with baseline HCV RNA ≥ 5 log10IU/ml, alanine aminotransferase (ALT) ≤ 2.5× the upper limit of normal, and compensated liver disease were dosed. Sequential cohorts of 10 patients, randomized 8:2 (active:placebo), received 25, 50, 75, and 100 mg of IDX184 once daily for 3 days, with a 14-day follow-up. There were no safety-related treatment discontinuations or serious adverse events. The adverse events and laboratory abnormalities observed for IDX184- and placebo-treated patients were similar. At the end of the 3-day treatment period, changes from baseline in HCV RNA levels (means ± standard deviations) were −0.5 ± 0.6, −0.7 ± 0.2, −0.6 ± 0.3, and −0.7 ± 0.5 log10for the 25-, 50-, 75-, and 100-mg doses, respectively, while viral load remained unchanged for the pooled placebo patients (−0.05 ± 0.3 log10). Patients with genotype-1a and patients with genotype-1b responded similarly. Serum ALT levels decreased, especially at daily doses ≥ 75 mg. During the posttreatment period, plasma viremia and serum aminotransferase levels returned to near pretreatment levels. No resistance mutations associated with IDX184 were detected. Plasma exposure of IDX184 and its nucleoside metabolite 2′-MeG was dose related and low. Changes in plasma viral load correlated with plasma exposure of 2′-MeG. In conclusion, the results from this proof-of-concept study show that small doses of the liver-targeted prodrug IDX184 were able to deliver significant antiviral activity and support further clinical evaluation of the drug candidate.

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.

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