| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Among 7 hepatitis C virus (HCV) proteins(core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), only NS5B, a viral RNA-dependent RNA polymerase, activated the interferon (IFN)-beta promoter However, mutant NS5B without RNA dependent RNA polymerase activity of template/primer association did not activate the IFN-beta promoter. Activation of the IFN-beta promoter by NS5Brequired the positive regulatory domain III, a binding sequence for IFN regulatory factor (IRF)-3. Moreover IRF-3 was phosphorylated by NS5B. Both inhibition of Toll-like receptor (TLR)3 expression by small interfering RNA and expression of the dominant negative form of Toll/IL-1 receptor domain- containing adapter inducing IFN-beta (TRIF) significantly reduced NS5B-induced activation of IFN-beta. Of the six other HCV proteins, NS4A, NS4B, and NS5A efficiently inhibited this activation. HCV NS5B is a potent activator of the host innate immune system, possibly through TLR3/TRIF and synthesis of dsRNA Meanwhile, NS4A, NS4B, and NS5A
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block IFN-beta induction by NS5B, which may contribute toward the persistence of this virus.
p53 could have a crucial role in the cellular innate defense against HCV. Significantly higher levels of HCV RNA replication and viral protein expression in the Huh7 cells were observed when their p53 expressions were knocked down. Moreover, IFN treatment was less effective in inhibiting the HCV RNA replication in the p53-knocked-down (p53kd) Huh7 cells. In fact, the activation of the IFN-stimulated response elements (ISRE) and the induction of IFN-stimulated genes (ISGs) were significantly attenuated in the p53kd Huh7 cells and p53 was found to directly interact with IFN regulatory factor (IRF)9. These observations underscore the potential contributions of the tumor suppresser p53 in cellular antiviral immunity against HCV with possible therapeutic implications.
Double-stranded-RNA activated protein kinase (PKR) is one of ISGs. We established PKR knockdown Huh7 cells using RNA interference and investigated the effect of PKR against HCV replication. In stable PKRkd cells, HCV replication was higher than that of control cells. Furthermore, stable PKRkd cells secreted significantly more HCV particles than did control cells. The replication of HCV was suppressed by the addition of IFN-alpha in both cells, and even 10 U/ml of IFN-alpha suppressed the replication of HCV more than 98% in both cells. PKR plays an important role in suppressing HCV replication in an innate state, but is not essential in IFN therapy.
2'-5'-oligoadenylate syntetase 1(OAS-1) is one of ISGs. We evaluated single nucleotide polymorphisms (SNPs) of OAS-1 and its relationship with stage of chronic HCV infection. 6 SNPs of OAS-1 were selected and examined in 409 Japanese patients with chronic HCV infection. Patients with genotypes A/A, A/G, and G//G of a SNP of OAS-1 at the exon 3 of its coding sequence were at gradient increased risks of suffering from higher serum alanine aminotransferase, higher degree of liver fibrosis, and higher presence of liver cirrhosis. Moreover, OAS-1 with G allele showed lower ability of inhibiting virus replication compared to OAS-1 with A allele. In conclusion, the SNP of OAS-1 at the exon 3 of its coding sequence was associated with progression of disease in Japanese patients with HCV infection. Less
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