Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity.
Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity.
复制标题
DOI：
10.1016/j.virol.2007.09.019
复制
发表时间：
2008-02
期刊：
影响因子：
3.7
通讯作者：
Yuko Sekine‐Osajima;N. Sakamoto;Kako Mishima;M. Nakagawa;Yasuhiro Itsui;M. Tasaka;Yuki Nishimura‐Sakurai;Cheng-Hsin Chen;T. Kanai;K. Tsuchiya;T. Wakita;N. Enomoto;Mamoru Watanabe
中科院分区：
文献类型：
--
作者：
Yuko Sekine‐Osajima;N. Sakamoto;Kako Mishima;M. Nakagawa;Yasuhiro Itsui;M. Tasaka;Yuki Nishimura‐Sakurai;Cheng-Hsin Chen;T. Kanai;K. Tsuchiya;T. Wakita;N. Enomoto;Mamoru Watanabe
HCV体外培养可导致大量细胞死亡，这表明存在HCV诱导的细胞病变效应。因此，我们利用HCV- jfh1细胞培养和新开发的HCV斑块测定技术来研究其机制和参与这种效应的病毒核苷酸序列。根据接种物滴度的不同，斑块试验产生了细胞病变斑块。在病毒感染的细胞中，内质网应激标志物GRP78和磷酸化的eif2 - α过表达。斑块中的细胞对凋亡标志物膜联蛋白v呈强烈阳性，从单个斑块中分离出的病毒亚克隆比亲本病毒具有更高的复制效率和细胞致病性。经空斑纯化的病毒有9个氨基酸取代，其中5个聚集在NS5B区的C端。综上所述，HCV感染的细胞病变效应涉及内质网应激诱导的细胞凋亡。某些HCV基因组结构可能决定病毒复制能力和细胞致病性。
HCV culture in vitro results in massive cell death, which suggests the presence of HCV-induced cytopathic effects. Therefore, we investigated its mechanisms and viral nucleotide sequences involved in this effect using HCV-JFH1 cell culture and a newly developed HCV plaque assay technique. The plaque assay developed cytopathic plaques, depending on the titer of the inoculum. In the virus-infected cells, the ER stress markers, GRP78 and phosphorylated eIF2-alpha, were overexpressed. Cells in the plaques were strongly positive for an apoptosis marker, annexin V. Isolated virus subclones from individual plaque showed greater replication efficiency and cytopathogenicity than the parental virus. The plaque-purified virus had 9 amino acid substitutions, of which 5 were clustered in the C terminal of the NS5B region. Taken together, the cytopathic effect of HCV infection involves ER-stress-induced apoptotic cell death. Certain HCV genomic structures may determine the viral replication capacity and cytopathogenicity.