Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses

Chuck, Chi-Pang; Chow, Hak-Fun; Wan, David Chi-Cheong; Wong, Kam-Bo

We have previously created a 1968 substrate library by performing saturation mutagenesis at P5 to P3' positions on the wild type (WT) sequence (SAVLQQSGF), which corresponds to the autocleavage sequence at the N-terminus of SARS-CoV 3CL pro [26] . The values of k obs /[3CL pro ] of the proteases against this WT sequence were 443611, 124613, 18065 and 174619 mM -1 min -1 for HCoV-NL63 (group 1), HCoV-OC43 (group 2a), SARS-CoV (group 2b), and IBV (group 3), respectively. That all proteases can cleave the WT sequence efficiently justifies that we can use our substrate library to profile the substrate specificities of 3CL pro from other groups of CoVs. Based on the FRET assay we developed, we measured the activities of 3CL pro from HCoV-NL63, HCoV-OC43, SARS-CoV and IBV against the 1968 substrate variants ( Figure 1 , Table S1 ) [26] . To identify the structural basis of substrate preferences for different CoVs, the protease activities were correlated with side chain volume [28] , hydrophobicity [29] , and a-helix and b-sheet propensities [30] as described [26] . The correlations were quantified in terms of correlation coefficients and p-values ( Figure 2 , Table S2 ).