Targett-Adams Paul, McLauchlan John
MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK.
J Gen Virol. 2005 Nov;86(Pt 11):3075-3080. doi: 10.1099/vir.0.81334-0.
Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.
构建了双顺反子亚基因组丙型肝炎病毒（HCV）复制子，其包含来自2a基因型毒株JFH1的序列，该序列还在HCV内部核糖体进入位点元件的控制下整合了萤火虫荧光素酶基因。在72小时内监测含有体外转录的亚基因组JFH1 RNA的Huh-7细胞提取物中的荧光素酶活性，以检查在没有任何选择压力的情况下HCV复制的早期阶段。复制子产生的酶活性比相应的1b基因型复制子产生的酶活性高近200倍，并且与NS5A蛋白和复制子RNA的积累相关。瞬时复制对IFN治疗呈剂量依赖性敏感，并且除了Huh-7细胞外，U2OS人骨肉瘤细胞系也支持JFH1复制子的有效复制。因此，这个基于JFH1序列的系统比先前的1b基因型复制子在病毒RNA复制的定量测量方面有改进。
