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Establishment of an indicator cell system for hepatitis C virus
Author(s) -
Tanaka Yoshinori,
Mori Yoshio,
Tani Hideki,
Abe Takayuki,
Moriishi Kohji,
Kojima Hirotatsu,
Nagano Tetsuo,
Okabe Takayoshi,
Suzuki Tetsuro,
Tatsumi Masashi,
Matsuura Yoshiharu
Publication year - 2010
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2010.00209.x
Subject(s) - ns2 3 protease , biology , hepatitis c virus , internal ribosome entry site , virology , ns3 , microbiology and biotechnology , luciferase , transcription factor , transcription (linguistics) , reporter gene , virus , promoter , viral replication , cell culture , gene expression , gene , messenger rna , transfection , biochemistry , translation (biology) , genetics , linguistics , philosophy
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.