Establishment of an indicator cell system for hepatitis C virus
Establishment of an indicator cell system for hepatitis C virus
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DOI：
10.1111/j.1348-0421.2010.00209.x
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发表时间：
2010-04
影响因子：
2.6
通讯作者：
Yoshinori Tanaka;Y. Mori;H. Tani;T. Abe;K. Moriishi;H. Kojima;T. Nagano;T. Okabe;Tetsuro Suzuki;M. Tatsumi;Y. Matsuura
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文献类型：
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作者：
Yoshinori Tanaka;Y. Mori;H. Tani;T. Abe;K. Moriishi;H. Kojima;T. Nagano;T. Okabe;Tetsuro Suzuki;M. Tatsumi;Y. Matsuura
尽管已开发出HCV JFH-1株的细胞培养系统，但尚无适用于血清源性HCV的稳健细胞培养系统。在这项研究中，我们建立了能够监测JFH-1病毒感染的系统，该系统基于特异性报告基因表达，通过HCV NS 3/4A蛋白酶对嵌合转录因子进行蛋白水解。我们利用了一种转录因子Gal 4-TBP，其协同增强GAL 4 UAS和HIV-1 LTR串联启动子与达特蛋白的转录。我们构建了嵌合达特和Gal 4-TBP转录因子，它们含有线粒体驻留IPS-1的HCV NS 3/4A切割序列，但不含有HCV多聚蛋白的切割序列，并操纵它们定位于ER。在感染JFH-1病毒后，转录因子被HCV蛋白酶有效切割，迁移到细胞核中并激活串联启动子下的报告基因。在感染JFH-1病毒后，携带转录因子和启动子下的荧光素酶基因的Huh 7 OK 1/TG-Luc细胞系以剂量依赖性方式表达荧光素酶，与HCV RNA复制密切相关。在用JFH-1病毒感染后，通过免疫磁性细胞分选选择性地浓缩携带转录因子和在启动子下的人低亲和力神经生长因子受体的cDNA的Huh 7 OK 1/TG-LNGFR细胞。这些结果表明，携带ER-驻留IPS-1序列的嵌合构建体被HCV蛋白酶特异性识别和有效切割，并用于检测HCV复制和回收HCV感染的细胞。该策略可能适用于建立用于分离血清源性HCV的细胞培养系统。
Although a cell culture system for HCV JFH‐1 strain has been developed, no robust cell culture system for serum‐derived HCV is available. In this study, we have established systems capable of monitoring infection with JFH‐1 virus based on specific reporter gene expression through proteolysis of chimeric transcription factors by HCV NS3/4A protease. We utilized a transcriptional factor Gal4‐TBP that synergistically enhances transcription of the GAL4UAS and HIV‐1 LTR tandem promoter with the Tat protein. We constructed chimeric Tat and Gal4‐TBP transcription factors containing the HCV NS3/4A cleavage sequence of a mitochondria‐resident IPS‐1, but not those of the HCV polyprotein, and manipulated them to localize in the ER. Upon infection with JFH‐1 virus, the transcription factors were efficiently cleaved by HCV protease, migrated into the nucleus and activated the reporter gene under the tandem promoter. Upon infection with JFH‐1 virus, the Huh7OK1/TG‐Luc cell line carrying the transcription factors and a luciferase gene under the promoter expressed luciferase in a dose‐dependent manner in close correlation with HCV RNA replication. Huh7OK1/TG‐LNGFR cells carrying the transcription factors and a cDNA of human low affinity nerve growth factor receptor under the promoter were selectively concentrated by immunomagnetic cell sorting upon infection with JFH‐1 virus. These results indicate that the chimeric constructs bearing the ER‐resident IPS‐1 sequence are specifically recognized and efficiently cleaved by HCV protease and are harnessed for detection of HCV replication and for recovery of HCV‐infected cells. This strategy may be applicable for the establishment of cell culture systems for the isolation of serum‐derived HCV.