A mammalian cell-based reverse two-hybrid system for functional analysis of 3C viral protease of human enterovirus 71

Analytical Biochemistry
Jin-Ching LeeJohn T A Hsu

Abstract

Although several cell-based reporter assays have been developed for screening of viral protease inhibitors, most of these assays have a significant limitation in that numerous false positives can be generated for the compounds that are interfering with reporter gene detection due to the cellular viability. To improve, we developed a mammalian cell-based assay based on the reverse two-hybrid system to monitor the proteolytic activity of human enterovirus 71 (EV71) 3C protease and to validate the cytotoxicity of compounds at the same time. In this system, the GAL4 DNA binding domain (M3) and transactivation domain (VP16) were fused, in-frame, with 3C or 3C(mut). The 3C(mut) was an inactivated protease with mutations at the predicted catalytic triad. The reporter plasmid contains a secreted alkaline phosphatase (SEAP) gene under the control of GAL4 activating sequences. We demonstrated that M3-3C-VP16 failed to turn on the expression of SEAP due to the separation of M3 and the VP16 domains by self-cleavage of 3C. In contrast, SEAP expression was induced by the M3-3C(mut)-VP16 fusion protein or the M3-3C-VP16 in cells treated with AG7088, a potent inhibitor of human rhinoviruses (HRVs) 3C protease. Potentially, this protease detect...Continue Reading

References

May 1, 1992·Proceedings of the National Academy of Sciences of the United States of America·B DasmahapatraJ Schwartz
Sep 17, 1996·Proceedings of the National Academy of Sciences of the United States of America·M VidalJ D Boeke
Sep 25, 1999·The New England Journal of Medicine·M HoS R Shih
Sep 29, 1999·Proceedings of the National Academy of Sciences of the United States of America·D A MatthewsS T Worland
Jan 20, 2000·Virology·A BarcoL Carrasco
May 18, 2000·Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology·C C LiuI J Su
Oct 6, 2000·Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America·L G ChanL J Anderson
Aug 15, 2001·Antimicrobial Agents and Chemotherapy·K LindstenN P Dantuma
Oct 2, 2002·The Journal of Infectious Diseases·Tzou-Yien LinCheng-Hsun Chiu
Jan 22, 2005·The Journal of General Virology·M Steven ObersteMark A Pallansch
May 27, 2005·Antiviral Research·Tsui-Ying WengTrai-Ming Yeh
Sep 27, 2005·Assay and Drug Development Technologies·Jin-Ching LeeJohn T-A Hsu

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Citations

May 4, 2011·The Journal of Infectious Diseases·Hui-Chen HungJohn T-A Hsu
Oct 22, 2008·The Journal of Antimicrobial Chemotherapy·Tzu-Chun ChenShin-Ru Shih
Jan 22, 2010·The Journal of Antimicrobial Chemotherapy·Hui-Chen HungChing-Ping Tseng
Jan 24, 2013·Virology Journal·Rei-Lin Kuo, Shin-Ru Shih
Jan 13, 2010·World Journal of Gastroenterology : WJG·Xiao-Nan ZhangZheng-Hong Yuan
Jan 8, 2014·Antiviral Research·Lonneke van der LindenFrank J M van Kuppeveld
Aug 13, 2015·Viruses·Lonneke van der LindenFrank J M van Kuppeveld
Dec 25, 2012·Antiviral Research·Luqing ShangZheng Yin
Dec 16, 2018·Virologica Sinica·Huiqiang Wang, Yuhuan Li
Jun 3, 2021·Bioorganic & Medicinal Chemistry·Yaping ChengXiaoqing Cai

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