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A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
Binding interaction of SARS coronavirus 3CL(pro) protease with vacuolar-H+ ATPase G1 subunit
Cheng-Wen Lin1, Fuu-Jen Tsai, Lei Wan
1Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan. cwlin@mail.cmu.edu.tw
Abstract:
The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important issue for treatment and prevention of SARS. Recently, SARS-CoV 3CL(pro) protease has been implied to be possible relevance to SARS-CoV pathogenesis. In this study, we intended to identify potential 3CL(pro)-interacting cellular protein(s) using the phage-displayed human lung cDNA library. The vacuolar-H+ ATPase (V-ATPase) G1 subunit that contained a 3CL(pro) cleavage site-like motif was identified as a 3CL(pro)-interacting protein, as confirmed using the co-immunoprecipitation assay and the relative affinity assay. In addition, our result also demonstrated the cleavage of the V-ATPase G1 fusion protein and the immunoprecipitation of cellular V-ATPase G1 by the 3CL(pro). Moreover, loading cells with SNARF-1 pH-sensitive dye showed that the intracellular pH in 3CL(pro)-expressing cells was significantly lower as compared to mock cells.
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