Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system
Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system
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DOI：
10.1016/j.bbrc.2005.02.138
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发表时间：
2005-04-22
影响因子：
3.1
通讯作者：
Kato, N
中科院分区：
文献类型：
--
作者：
Ikeda, M;Abe, K;Kato, N
最近，我们报道了丙型肝炎病毒(丙型肝炎病毒)的一个亚基因组复制子，该复制子来源于丙型肝炎病毒(丙型肝炎病毒O株)，感染了非肿瘤性人肝细胞PH5CH8。在这项研究中，我们从丙型肝炎病毒O中开发了一个基因组长度的双顺反子丙型肝炎病毒RNA。用干扰素从克隆的亚基因组复制子细胞系(SO)中获得治愈的HH-7细胞系(SOC)，并将其用于基因组长度的丙型肝炎病毒RNA(HCVRNA)的转染。将基因组长度的丙型肝炎病毒RNA导入SOC细胞后，通过G418处理，成功地筛选出一株克隆的细胞系O，证实了丙型肝炎病毒RNA和蛋白的高效表达。用干扰素处理从克隆细胞(O)中获得了一株治愈的OC。当基因组长度的丙型肝炎病毒RNA被导入Oc细胞时，克隆数量急剧增加。然而，克隆的治愈细胞系Soc和Oc尽管起源相同，但在集落形成效率上存在差异。这一结果表明，即使是克隆的细胞系也可以在细胞培养过程中改变其特征。对O细胞中的丙型肝炎病毒RNA进行了序列分析，发现NS3解旋酶区域(K1609E)存在氨基酸替换。该替换在瞬时报告和克隆形成试验中作为适应性突变。利用这种适应性突变和Oc细胞在克隆形成方面的优势，我们建立了第一个编码荧光素酶基因的基因组长度的双顺反子丙型肝炎病毒RNA高效复制的细胞系。该培养体系为研究丙型肝炎病毒复制和大规模筛选抗丙型肝炎病毒试剂提供了有用的工具。(C)2005 Elsevier Inc.保留所有权利。
Recently we reported a subgenomic hepatitis C virus (HCV) replicon derived from HCV (HCV-O strain) infected in non-neoplastic human hepatocyte PH5CH8. In this study, we developed a genome-length dicistronic HCV RNA from HCV-O. A cured HuH-7 cell line (sOc) was obtained from a cloned subgenomic replicon cell line (sO) by interferon (IFN) treatment and used for transfection with genome-length HCV RNA. One cloned cell line, O, was successfully selected by G418 treatment following the introduction of genome-length HCV RNA into sOc cells, and the robust expression of HCV RNA and proteins was confirmed. Oc, a cured cell line, was also obtained from the cloned cell line (O) by IFN treatment. The number of colonies increased drastically when genome-length HCV RNA was introduced into Oc cells. However, the cloned cured cell lines, sOc and Oc, differed in their colony formation efficiency despite their common origin. This result suggests that even a cloned cell line can change its characteristics during cell culture. Sequence analysis of HCV RNA from the O cells revealed an amino acid substitution in the NS3 helicase region (K1609E). This substitution worked as an adaptive mutation in transient reporter and colony formation assays. Using the advantages of this adaptive mutation and of Oc cells in colony formation, we established the first cell line in which genome-length dicistronic HCV RNA encoding a luciferase gene replicated efficiently. This culture system is useful tool for the study of HCV replication and mass screening for anti-HCV reagents. (c) 2005 Elsevier Inc. All rights reserved.