| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
The main purpose of this research project is to reveal the mechanism of hepatitis C viral genome replication by analysis of molecular structure of the replication complex and the functions of its components, using the cultured cells, in which the subgenomic RNA of hepatitis C virus (HCV) is self-replicating and maintained efficiently, so called HCV subgenomic replicon bearing cells. At first, we established the cell lines maintaining the new replicon RNA with full length genome RNA of HCV. Several biochemical characterizations of the replication complex in those cells showed that structural and functional properties of the replication complex are quite similar to those observed in the subgenomic replicon bearing cells. We also constructed the replicon RNA which contains the firefly luciferase protein encoding sequence in order to detect the replication activity easily as luciferase activity. Using this reporter containing replicon RNA. we investigated the interaction between the replic
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ation complex and cellular factors through studies of the effects of several cytokines and chemical reagents on the replication of the replicon. Results were as follows ; Transforming growth factor beta was found to be a suppressive factor for the replication of the replicon, possibly through the inhibition of cell proliferation. PD98059, inhibitor of MAP kinase signaling pathway, enhanced the replication of replicon, possibly through the activation of internal ribosome entry site-dependent translation which drives the translation from the HCV genome. Moreover, we studied the mechanism of inhibition of HCV genome replication by cyclosporin A (CsA), which we previously reported as a candidate for new anti-HCV reagent. As the results, we found that a target molecule of CsA for this inhibition is cyclophilin B (CyPB). Then, we revealed that CyPB is one of candidates for cellular components of HCVgenomic replication complex and that CyPB functions on the enhancement of RNA binding property of NS5B, the viral RNA polymerase, through the mutural interaction. From these findings, we concluded that CsA suppresses HCV genomic replication through the inhibition of CyPB activity, suggesting that CyPB could be a candidate of the novel target for development of anti-HCV drugs. Less
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