Genetic screen for monitoring severe acute respiratory syndrome coronavirus 3C-like protease.
A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome. Site-specific proteolysis plays a critical role in regulating a number of cellular and viral processes. Since the main protease of SCoV, also termed 3C-like protease, is an attractive target for drug therapy, we have developed a safe, simple, and rapid genetic screen assay to monitor the activity of the SCoV 3C-like protease. This genetic system is based on the bacteriophage lambda regulatory circuit, in which the viral repressor cI is specifically cleaved to initiate the lysogenic-to-lytic switch. A specific target for the SCoV 3C-like protease, P1/P2 (SAVLQ/SGFRK), was inserted into the lambda phage cI repressor. The target specificity of the SCoV P1/P2 repressor was evaluated by coexpression of this repressor with a chemically synthesized SCoV 3C-like protease gene construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI. SCoV P1/P2-cro and the beta-galactosidase-SCoV 3C-like protease constructs, lambda phage replicated up to 2,000-fold more efficiently than in cells that did not express the SCoV 3C-like protease. This simple and highly specific assay can be used to monitor the activity of the SCoV 3C-like protease, and it has the potential to be used for screening specific inhibitors.
Parera
Mariona
M
Fundacio irsiCaixa, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain.
Clotet
Bonaventura
B
Martinez
Miguel Angel
MA
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005821:Genetic Techniques; D006801:Humans; D008969:Molecular Sequence Data; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D045169:Severe Acute Respiratory Syndrome / Q000821:virology*
D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases