Development of anti-HCV therapy the regulation of NS5A function

丙型肝炎病毒(丙型肝炎病毒)亚基因组复制子在人肝癌细胞Huh-7中持续高效复制。为了将先前的结果推广到其他分离的丙型肝炎病毒克隆，我们从黑猩猩感染的克隆之一HC-J4中构建了另一个丙型肝炎病毒复制子。丙型肝炎病毒复制子RpJ4由丙型肝炎病毒5‘-非编码区、新霉素磷酸转移酶基因、脑心肌炎病毒IRES、非结构区、NS3到NS5B和丙型肝炎病毒3’-非编码区组成。已知的丙型肝炎病毒-Con1复制子所需的适应性突变被引入到RpJ4复制子a.(氨基酸数量根据HC-J4)2197丝氨酸到脯氨酸，aa.2201丝氨酸缺失，aa.2204丝氨酸到异亮氨酸(RpJ4-S2I97P，RpJ4-S22001del，和RpJ4-S2204I)。在干扰素敏感性决定区(ISDR)引入6个氨基酸突变，构建了RpJ4/ISDR突变体和RpJ4-S2201del/ISDR突变体。将复制子RNA导入Huh-7细胞，经G418筛选获得稳定表达复制子的细胞系。RpJ4和RpJ4/ISDR突变株在转染初代Huh-7细胞后未产生G418抗性克隆。相反，RpJ4-S2197P、RpJ4-S2204I、RpJ4-S2201del和RpJ4-S2201del/ISDR突变体可以有效地转导G418抗性细胞，其中RpJ4-S2201del/ISDR突变体的转导效率最高。总之，在ISDR的上游区域引入适应性突变后，来自HC-J4的丙型肝炎病毒复制子可以有效地复制。此外，在ISDR中额外引入突变进一步增强了其复制能力。这些发现表明，NS5A结构域的遗传结构在丙型肝炎病毒1b复制中起关键作用。

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.