| Budget Amount *help |
¥11,700,000 (Direct Cost: ¥11,700,000)
Fiscal Year 2002: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2001: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2000: ¥4,100,000 (Direct Cost: ¥4,100,000)
|
|---|
| Research Abstract |
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 have constructed another HCV replicon from HC-J4, one of the chimpanzee-infectious clones. An HCV-replicon derived from HC-J4 (RpJ4) consists of HCV-5'-UTR, neomycin phosphotransferase gene, the encephalomyocarditis virus IRES, HCV-nonstructural region, NS3 to NS5B, and HCV-3'-UTR. The adaptive mutations known to be required for HCV-Conl 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-S2I97P, RpJ4-S22001del, and RpJ4-S2204I). RpJ4/ISDRmutant and RpJ4-S2201del/ISDRmutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). Replicon RNA was transfected into Huh-7 cells, and stable replicon-expressing cell lines were established by G418 selection. After transfection to naive Huh-7 cells, RpJ4 and RpJ4/ISDRmutants did not produce any G418-resistant colonies. In contrast, G418-resistant cells were transduced efficiently by RpJ4-S2197P, RpJ4-S2204I, RpJ4-S2201del and RpJ4-S2201del/ISDRmutants, with the RpJ4-S2201del/ISDRmutant being most efficient. In conclusion, 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 enhances its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV-1b replications.
|
