Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee‐infectious HC‐J4 isolate to replicate efficiently in Huh‐7 cells
Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee‐infectious HC‐J4 isolate to replicate efficiently in Huh‐7 cells
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DOI：
10.1111/j.1365-2893.2004.00525.x
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发表时间：
2004-09
影响因子：
2.5
通讯作者：
S. Maekawa;N. Enomoto;N. Sakamoto;M. Kurosaki;E. Ueda;T. Kohashi;H. Watanabe;C.‐H. Chen;T. Yamashiro;Y. Tanabe;N. Kanazawa;M. Nakagawa;C. Sato;M. Watanabe
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文献类型：
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作者：
S. Maekawa;N. Enomoto;N. Sakamoto;M. Kurosaki;E. Ueda;T. Kohashi;H. Watanabe;C.‐H. Chen;T. Yamashiro;Y. Tanabe;N. Kanazawa;M. Nakagawa;C. Sato;M. Watanabe
摘要据报道，丙型肝炎病毒（HCV）亚基因组复制子在人肝癌Huh-7细胞中有效且连续地复制。为了将先前的结果扩展到其他分离的HCV克隆，我们从黑猩猩感染性HCV克隆之一HC-J 4构建了另一个HCV复制子。HC-J 4衍生的HCV复制子（RpJ 4）由HCV-5′非翻译区、新霉素磷酸转移酶基因、脑心肌炎病毒内部核糖体进入位点、HCV非结构区、NS 3至NS 5 B和HCV-3′非翻译区组成。已知HCV-Con 1复制子所需的适应性突变被引入RpJ 4复制子aa。（根据HC-J 4的氨基酸编号）2197丝氨酸变为脯氨酸，aa. 2201处丝氨酸缺失，aa. 2204丝氨酸变为异亮氨酸（RpJ 4-S2197 P、RpJ 4-S22001 del和RpJ 4-S2204 I）。还通过在干扰素敏感性决定区（ISDR）引入6个氨基酸突变构建了RpJ 4/ISDR突变体和RpJ 4-S2201 del/ISDR突变体。转染Huh-7细胞和G418筛选后，RpJ 4和RpJ 4/ISDR突变体不产生任何集落。相反，G418抗性细胞被RpJ 4-S2197 P、RpJ 4-S2204 I、RpJ 4-S2201 del和RpJ 4-S2201 del/ISDR突变体有效地转导，其中RpJ 4-S2201 del/ISDR突变体最有效。因此，在ISDR上游区域引入适应性突变后，来自HC-J 4的HCV复制子可以有效复制。此外，向ISDR中额外引入突变进一步增强了其复制。这些发现表明NS 5A结构域的遗传结构在HCV复制中至关重要。
Summary. 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 constructed another HCV replicon from HC‐J4, one of chimpanzee‐infectious HCV clones. An HCV replicon derived from HC‐J4 (RpJ4) consists of HCV‐5′ untranslated region, neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural region, NS3 to NS5B, and HCV‐3′ untranslated region. The adaptive mutations known to be required for HCV‐Con1 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‐S2197P, RpJ4‐S22001del, and RpJ4‐S2204I). RpJ4/ISDR mutant and RpJ4‐S2201del/ISDR mutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). After transfection into Huh‐7 cells and G418 selection, RpJ4 and RpJ4/ISDR mutants did not produce any colony. In contrast, G418‐resistant cells were transduced efficiently by RpJ4‐S2197P, RpJ4‐S2204I, RpJ4‐S2201del and RpJ4‐S2201del/ISDR mutant, with the RpJ4‐S2201del/ISDR mutant being most efficient. Hence 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 enhanced its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV replications.