DETERMINANTS OF MOUSE HEPATITIS-VIRUS 3C-LIKE PROTEINASE ACTIVITY

The coronavirus, mouse hepatitis virus strain A59 (MHV), expresses a c hymotrypsin-like cysteine proteinase (3CLpro) within the gene 1 polypr otein. The MHV 3CLpro is similar to the picornavirus 3C proteinases in the relative location of confirmed catalytic histidine and cysteine r esidues and in the predicted use of Q/(S, A, G) dipeptide cleavage sit es. However, less is known concerning the participation of aspartic ac id or glutamic acid residues in catalysis by the coronavirus 3C-like p roteinases or of the precise coding sequence of 3CLpro within the gene 1 polyprotein. In this study, aspartic acid residues in MHV 3CLpro we re mutated and the mutant proteinases were tested for activity in an i n vitro trans cleavage assay. MHV 3CLpro was not inactivated by substi tutions at Asp(3386) (D53) or Asp(3398) (D65), demonstrating that they were not catalytic residues. MHV 3CLpro was able to cleave at a gluta mine-glycine (QG(3607-8)) dipeptide within the 3CLpro domain upstream from the predicted carboxy-terminal QS(3635-6) cleavage site of 3CLpro . The predicted full-length 3CLpro (S-3334 to Q(3635)) had an apparent mass of 27 kDa, identical to the p27 3CLpro in cells, whereas the tru ncated proteinase (S-3334 to Q(3607)) had an apparent mass of 24 kDa. This 28-amino-acid carboxy-terminal truncation of 3CLpro rendered it i nactive in a trans cleavage assay. Thus, MHV 3CLpro was able to cleave at a site within the putative full-length proteinase, but the entire predicted 3CLpro domain was required for activity. These studies sugge st that the coronavirus 3CL-proteinases may have a substantially diffe rent structure and catalytic mechanism than other 3C-like proteinases. (C) 1997 Academic Press.