INTRACELLULAR AND IN VITRO-TRANSLATED 27-KDA PROTEINS CONTAIN THE 3C-LIKE PROTEINASE ACTIVITY OF THE CORONAVIRUS MHV-A59

The coronavirus mouse hepatitis virus-A59 (MHV-A59) encodes a serine-l ike proteinase (3C-like proteinase or 3CLpro) in ORF la of gene 1 betw een nucleotides 10209 and 11114. We previously have demonstrated that proteins expressed in vitro from a cDNA clone of the 3CLpro region pos sess proteinase activity, and that the proteinase is able to cleave su bstrate in trans. We sought to determine if the 27-kDa in vitro cleava ge product (p27) was an active form of the 3CLpro and whether this was consistent with the 3CLpro expressed in virus-infected cells. Antibod ies directed against the 3CLpro domain detected 27-kDa MHV proteins in vitro and in MHV-A5g-infected cells. The 27-kDa proteins were able to cleave substrate in trans without other protein cofactors or suppleme ntal membranes, and the p27 proteinase activity was retained after pur ification by immunoprecipitation and gel electrophoresis. When p27 was expressed in vitro with portions of the amino- and carboxy-terminal f lanking domains (MPI and MP2), p27 was not liberated by cis cleavage. The proteolytic activity of the 27-kDa proteins was inhibited by a var iety of cysteine and serine proteinase inhibitors, and was eliminated by the cysteine proteinase inhibitor E64d. These results indicate that the 27-kDa protein is a mature proteinase in MHV-A59-infected cells, and that appropriate processing of this molecule occurs in vitro. (C) 1996 Academic Press, Inc.