CHARACTERIZATION OF THE LEADER PAPAIN-LIKE PROTEINASE OF MHV-A59 - IDENTIFICATION OF A NEW IN-VITRO CLEAVAGE SITE

Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) g enome predicts the presence of two papain-like proteinases encoded wit hin the first open reading frame (ORF 1a) of the replicase gene. The m ore 5' of these domains, the leader papain-like proteinase, is respons ible for the cleavage of the amino terminal protein, p28. The core of this proteinase domain was defined to between amino acids 1084 and 131 6 from the beginning of ORF la. Through the use of deletion analysis c oupled with in vitro expression, we studied the role of the coding reg ion between p28 and the leader papain-like proteinase on the cleavage of p28 itself. Expression of a series of deletion mutants showed proce ssing of p28, albeit at lower levels. Reduced p28 production resulting from a 0.4-kb deletion positioned between p28 and the proteinase doma in suggests an involvement of this region in catalytic processing. Som e mutants displayed cleavage patterns indicative of a second cleavage site. Interestingly, this new cleavage site identified in vitro maps t o a position similar to the expected cleavage site of a p65 polypeptid e detected in MHV-A69-infected cells. Mutagenesis of the catalytic His 1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like proteinase encoded in ORF 1a. (C) 1995 Academi c Press, Inc.