CHARACTERIZATION OF A 2ND CLEAVAGE SITE AND DEMONSTRATION OF ACTIVITYIN TRANS BY THE PAPAIN-LIKE PROTEINASE OF THE MURINE CORONAVIRUS MOUSE HEPATITIS-VIRUS STRAIN A59

The 21.7-kb replicase locus of mouse hepatitis virus strain A59 (MHV-A 59) encodes several putative functional domains, including three prote inase domains. Encoded closest to the 5' terminus of this locus is the first papain-like proteinase (PLP-1) (S. C. Baker et al., J. Virol. 6 7:6056-6063, 1993; H.-J. Lee et al., Virology 180:567-582, 1991). This cysteine proteinase is responsible for the in vitro cleavage of p28, a polypeptide that is also present in MHV-A59-infected cells. Cleavage at a second site was recently reported for this proteinase (P. J. Bon illa et al., Virology 209:489-497, 1995). This new cleavage site maps to the same region as the predicted site of the C terminus of p65, a v iral polypeptide detected in infected cells. In this study, microseque ncing analysis of the radiolabeled downstream cleavage product and del etion mutagenesis analysis were used to identify the scissile bond of the second cleavage site to between Ala832 and Gly833. The effects of mutations between the P5 and P2' positions on the processing at the se cond cleavage site were analyzed. Most substitutions at the P4, P3, P2 , and P2' positions were permissive for cleavage, With the exceptions of a conservative P1 mutation, Ala832Gly and a conservative P5 mutatio n, Arg828Lys, substitutions at the P5, P1, and P1' positions severely diminished second-site proteolysis. Mutants in which the p28 cleavage site (Gly247 down arrow Val248) was replaced by the Ala832 down arrow Gly833 cleavage site and vice versa were found to retain processing ac tivity. Contrary to previous reports, me determined that the PLP-1 has the ability to process in trans at either the p28 site or both cleava ge sites, depending on the choice of substrate, The results from this study suggest a greater role by the PLP-1 in the processing of the rep licase locus in vivo.