China-Japan Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100101, China.
J Virol Methods. 2010 Nov;169(2):380-4. doi: 10.1016/j.jviromet.2010.08.009. Epub 2010 Aug 14.
Replication of infectious hepatitis C virus in Huh7 cells, a human hepatocyte cell line, has become possible due to the unique properties of the JFH1 isolate. Developing reporter virus systems for a simple titration has been attempted by integrating heterologous reporter genes into the JFH1 genome, resulting in a big infectivity reduction that limits the usefulness of such reporter systems. To overcome this problem, JFH1-infected Huh7 cells were cultured continuously for 2 years to obtain Huh7-adapted JFH1 variants capable of yielding up to 1000-fold higher titers. Sequence analysis of variant genome RNA suggested that this adapted population consisted mainly of two variants. By joining the 5'-half of the obtained representative viral complementary DNA (cDNA) fragments of the variants with the 3'-half of the wild-type's, two prototype clones, A/WT and B/WT, were constructed. Replication of A/WT and B/WT viruses in Huh7 cells showed up to 100-1000-fold higher titers than the wild-type. A Renilla luciferase cDNA was inserted into the Nonstructural Protein 5A region of the A/WT and B/WT cDNA to generate A/WT-Rluc and B/WT-Rluc, respectively. Transfection of Huh7 cells with in vitro-transcribed A/WT-Rluc and B/WT-Rluc RNA resulted in production of infectious viruses with approximately 15- and 25-fold higher titers, respectively, than the wild-type RNA. The replication of A/WT-Rluc and B/WT-Rluc viruses was more vigorous than the wild-type even with insertion of the luciferase cDNA showing a good correlation of luciferase activities with infectious titers. Furthermore, interferon-alpha inhibited the replication of A/WT-Rluc and B/WT-Rluc viruses in a dose-dependent manner as determined by a luciferase assay. These results imply that our system is potentially a tool useful for screening anti-hepatitis C virus drugs in a simple and time/cost-saving manner.
由于 JFH1 分离株的独特特性，使得在 Huh7 细胞（一种人肝细胞系）中复制传染性丙型肝炎病毒成为可能。通过将异源报告基因整合到 JFH1 基因组中，尝试开发用于简单滴定的报告病毒系统，但这会导致感染性大大降低，限制了此类报告系统的实用性。为了克服这个问题，JFH1 感染的 Huh7 细胞连续培养了 2 年，以获得能够产生高达 1000 倍更高滴度的适应 Huh7 细胞的 JFH1 变异体。变异体基因组 RNA 的序列分析表明，该适应群体主要由两种变异体组成。通过将获得的代表性病毒互补 DNA (cDNA) 片段的 5' 半部分与野生型的 3' 半部分连接，构建了两个原型克隆 A/WT 和 B/WT。A/WT 和 B/WT 病毒在 Huh7 细胞中的复制显示出比野生型高出 100-1000 倍的滴度。将 Renilla 荧光素酶 cDNA 插入 A/WT 和 B/WT cDNA 的非结构蛋白 5A 区域，分别生成 A/WT-Rluc 和 B/WT-Rluc。用体外转录的 A/WT-Rluc 和 B/WT-Rluc RNA 转染 Huh7 细胞，分别产生比野生型 RNA 高出约 15 倍和 25 倍的感染性病毒。与插入荧光素酶 cDNA 相比，A/WT-Rluc 和 B/WT-Rluc 病毒的复制更加旺盛，荧光素酶活性与感染滴度呈良好相关性。此外，干扰素-α通过荧光素酶测定以剂量依赖的方式抑制 A/WT-Rluc 和 B/WT-Rluc 病毒的复制。这些结果表明，我们的系统有可能成为一种有用的工具，用于以简单、省时和节省成本的方式筛选抗丙型肝炎病毒药物。
