Production of Infectious Hepatitis C Virus by Using RNA Polymerase I-Mediated Transcription
Production of Infectious Hepatitis C Virus by Using RNA Polymerase I-Mediated Transcription
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DOI：
10.1128/jvi.02397-09
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发表时间：
2010-06-01
影响因子：
5.4
通讯作者：
Suzuki, Tetsuro
中科院分区：
文献类型：
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作者：
Masaki, Takahiro;Suzuki, Ryosuke;Suzuki, Tetsuro
在这项研究中，我们使用RNA聚合酶I(POL I)转录系统来开发反向遗传学方案来产生丙型肝炎病毒(HCV)，这是一种无帽正链RNA病毒。用含有丙型肝炎病毒JFH-1全长c DNA的质粒，在Pol I启动子和Pol I终止子的两侧，产生一个两端没有额外序列的未剪接的RNA，导致在细胞质内有效的RNA复制和随后的感染性病毒粒子的产生。利用这项技术，我们开发了一种简单的复制子反式包装系统，在该系统中，通过瞬时转染两个质粒，可以将病毒基因组复制和病毒粒子组装作为两个独立的步骤进行检查。此外，我们建立了一个稳定的细胞系，该细胞系能够以较低的病毒基因组突变频率构成地产生丙型肝炎病毒。利用该细胞系评价了N-糖基化抑制剂对丙型肝炎病毒产生的影响，结果表明，病毒粒子组装、细胞内转运和分泌等某些步骤(S)可能根据丙型肝炎病毒包膜蛋白糖链的修饰而上调和下调。这种基于Pol I的丙型肝炎病毒表达系统将有利于高通量的抗病毒筛选和疫苗发现计划。
In this study, we used an RNA polymerase I (Pol I) transcription system for development of a reverse genetics protocol to produce hepatitis C virus (HCV), which is an uncapped positive-strand RNA virus. Transfection with a plasmid harboring HCV JFH-1 full-length cDNA flanked by a Pol I promoter and Pol I terminator yielded an unspliced RNA with no additional sequences at either end, resulting in efficient RNA replication within the cytoplasm and subsequent production of infectious virions. Using this technology, we developed a simple replicon trans-packaging system, in which transient transfection of two plasmids enables examination of viral genome replication and virion assembly as two separate steps. In addition, we established a stable cell line that constitutively produces HCV with a low mutation frequency of the viral genome. The effects of inhibitors of N-linked glycosylation on HCV production were evaluated using this cell line, and the results suggest that certain step(s), such as virion assembly, intracellular trafficking, and secretion, are potentially up- and downregulated according to modifications of HCV envelope protein glycans. This Pol I-based HCV expression system will be beneficial for a high-throughput antiviral screening and vaccine discovery programs.