| Budget Amount *help |
¥17,200,000 (Direct Cost: ¥15,700,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2007: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2006: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 2005: ¥5,700,000 (Direct Cost: ¥5,700,000)
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| Research Abstract |
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 sequence
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s 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.
The intracellular double-stranded (ds)RNA-mediated signal to induce type I interferon (IFN) plays a pivotal role in viral regulation. In hepatitis C virus (HCV), NS3/4A has been reported to block this system by cleaving IPS-1. However, an overall picture is still unclear, and it is unknown how different HCV strains affect this signal. Using three Huh7 cell lines (naive, replicon-harboring, and replicon-cured cells), poly I: C transfection-stimulated IRF-3 dimerization, and ISRE-promoter activation following overexpression of each signaling molecule were analyzed. Responsible HCV elements to block this signal were also investigated. To assess the role of HCV strain in the signal, the signal blocking capacities of HCV-N and HC-.J4 were compared. In contrast to naive and cured cells, IRF-3 dimerization was strongly suppressed in replicon-harboring cells, demonstrating HCV itself blocks the signal ISRE-promoter activation by RIG-I, and IPS-1 was remarkably suppressed in replicon-harboring cells, indicating RIG-I and/or IPS-1 were the main targets of HCV in this signal blockade. HCV-N-derived NS3/4A suppressed ISRE-promoter activation by RIG-I and IPS-I. HCV-N-derived NS4B also suppressed ISRE-promoter activation by IPS-I. On the other hand, HC-34-derived NS3/4A did not suppress ISRE-promoter activation by RIG-I and IPS-1. HCV replicon derived from HCV-N inhibits the dsRNA-mediated type I.
IFN induction signal through targeting RIG-I and/or IPS-1 by NS3/4A, and partly by NS4B. However, NS3/4A derived from HC-I4 could not significantly block the signal, indicating that the strain-specific blocking capacity might contribute to different replication capacity specific to each HCV replicon.
Recently, microdomains of organelle membranes rich in sphingomyelin and cholesterol (called "lipid rafts") have been considered to act as a scaffold for the hepatitis C virus(HCV) replication complex. Using the HCV cell culture system, we investigated the effect of myriocin, a sphingomyelin synthesis inhibitor, on HCV replication. We also investigated the combined effect of myriocin with interferon (IFN) and myriocin with simvastatin. Myriocin suppressed replication of both a genotype 1b subgenomic HCV replicon (Huh7/Rep-Feo) and genotype 2a infectious HCV(JFH-1 HCV) in a dose-dependent manner(for subgenomic HCV-1b, maximum of 79% at 1000nmol/L; for genomic HCV-2α, maximum of 40% at 1000 nmol/L). Combination treatment with myriocin and IFN or myriocin and simvastatin attenuated HCV RNA replication synergistically in Huh7/Rep-Feo cells. Our data demonstrate that the sphingomyelin synthesis inhibitor strongly suppresses replication of both the subgenomic HCV-1b replicon and the JFH-1 strain of genotype 2a infectious HCV, indicating that lipid metabolism could be a novel target for HCV therapy. Less
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