g-Hsin Chen

Synergistic Inhibition of Intracellular Hepatitis C Virus Replication by Combination of Ribavirin and Interferon-α

Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.

Regulation of Hepatitis C Virus Replication by Interferon Regulatory Factor 1

ABSTRACT Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the interferon-mediated antiviral system in cells expressing an HCV replicon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor 1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replicon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replicon. Moreover, in cured Huh7 cells from which the HCV replicon had been eliminated, the expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of interferon-stimulated-gene-mediated antiviral responses.

Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication.

Summary.  The number of amino acid substitutions in the interferon sensitivity‐determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon (IFN) response and viral load. Several HCV replicon‐based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear as to how different ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in the colony formation assay using HCV replicons with few mutations (0, 1 and 3) in the ISDR, numerous colonies (>200) appeared when using constructs with six mutations. Introduction of various distinct ISDR sequences with multiple mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically affected replication, including codon 2209 which, in patients, was closely associated with a strong response to IFN. ISDR sequences associated with a clinical IFN response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.

Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee-infectious HC-J4 isolate to replicate efficiently in Huh-7 cells.

Summary.  Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh‐7 cells. To extend the previous results to other isolated HCV clones, we constructed another HCV replicon from HC‐J4, one of chimpanzee‐infectious HCV clones. An HCV replicon derived from HC‐J4 (RpJ4) consists of HCV‐5′ untranslated region, neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural region, NS3 to NS5B, and HCV‐3′ untranslated region. The adaptive mutations known to be required for HCV‐Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC‐J4) 2197 serine to proline, deletion of serine at aa.2201, and aa.2204 serine to isoleucine (RpJ4‐S2197P, RpJ4‐S22001del, and RpJ4‐S2204I). RpJ4/ISDR mutant and RpJ4‐S2201del/ISDR mutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). After transfection into Huh‐7 cells and G418 selection, RpJ4 and RpJ4/ISDR mutants did not produce any colony. In contrast, G418‐resistant cells were transduced efficiently by RpJ4‐S2197P, RpJ4‐S2204I, RpJ4‐S2201del and RpJ4‐S2201del/ISDR mutant, with the RpJ4‐S2201del/ISDR mutant being most efficient. Hence the HCV replicon derived from HC‐J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR. Moreover, additional introduction of mutations into ISDR further enhanced its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV replications.

Overexpression of interferon γ -inducible protein 10 in the liver of patients with type i autoimmune hepatitis identified by suppression subtractive hybridization

OBJECTIVE:To clarify gene expression profiles in the liver may elucidate the pathogenesis of type I autoimmune hepatitis (AIH). Using suppression subtractive hybridization (SSH), we identified genes overexpressed in the liver of AIH.METHODS:A small liver biopsy sample from a patient with definite AIH was available to be analyzed in our system. By mixing cDNA synthesized from this sample as a ‘tester’ and cDNA from a normal liver as a ‘driver,’ we subtracted cDNA to enrich genes overexpressed in AIH. After polymerase chain reaction (PCR) amplification and subcloning, we identified subtracted genes by sequencing 50 randomly selected clones.RESULTS:Only one cDNA fragment, which is identical to interferon inducible protein 10 (IP-10), was overexpressed by >10 times in the liver of AIH, as compared with control. We confirmed IP-10 overexpression in all eight patients with AIH by reverse transcription PCR. Immunohistochemical analysis demonstrated increased IP-10 expression in hepatocytes in the liver of AIH. Reverse transcription PCR analysis of 63 liver biopsy samples with various liver diseases revealed that IP-10 expression was significantly higher in AIH (p = 0.025) and chronic hepatitis C (p = 0.0043) than in other liver diseases. Interestingly, the amount of IP-10 mRNA expression was correlated with serum ALT values in AIH (p = 0.0006), but not in chronic hepatitis C (p = 0.43).CONCLUSION:These results indicate the IP-10 expression in the liver might be used as a preferential marker of AIH, and that IP-10 has some pathophysiological roles in the liver damage of AIH.

Negative regulation of intracellular hepatitis C virus replication by interferon regulatory factor 3

BackgroundInterferon regulatory factor (IRF)-3 plays an important role in initiating cellular interferon-stimulated gene-mediated antiviral responses. In the present study, we evaluated the effects of IRF-3 expression and activation on intracellular hepatitis C virus (HCV) replication using an HCV replicon system.MethodsAn HCV replicon was constructed that expressed a neomycin-selectable chimeric firefly luciferase reporter protein. A small interfering (si) RNA oligonucleotide directed against IRF-3 mRNA was designed and synthesized. A eukaryote expression plasmid vector was constructed that expressed IRF-3 mRNA under control of the cytomegalovirus early promoter/enhancer. To evaluate transcriptional activity of the interferon-stimulated genes, a reporter vector was used that expressed firefly luciferase under control of the interferon-stimulated response element (ISRE).ResultsThe baseline expression of IRF-3 did not significantly differ between cells with and without expression of the replicon. Transfection of an IRF-3 expression plasmid into the cells raised the ISRE-luciferase activities. The increase of ISRE activity was significantly more potent in the replicon-expressing cells than in cells without replicon expression. Concomitantly, the overexpression of IRF-3 suppressed HCV replication levels. In contrast, siRNA knockdown of IRF-3 suppressed ISRE activity by 38% ± 2%. Interestingly, the suppression of IRF-3 resulted in a significant increase of HCV replication, by up to twofold, depending on the IRF-3 suppression levels.ConclusionsIRF-3 negatively regulated intracellular HCV replication, and was partially activated in cells that expressed the HCV replicon. Thus, IRF-3 is a key molecule controlling HCV replication through modulation of host interferon gene responses.

Establishment of a New Hepatitis C Virus-1 b Replicon

Backgrounds:Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh7 cells (Lohmann, Science 1999). Their replications can be enhanced by introduction of certain cell culture-adaptive mutations in the viral NS5A region (Blight, Science 2000, Krieger, J Virol 2001). To extend the previous results to other isolated HCV clones, we have constructed another HCV replicon from HC-J4, one of the chimpanzee-infectious clones, and demonstrated efficient intracellular replications in Huh7 cells.