Pj. Bonilla et al., CHARACTERIZATION OF THE LEADER PAPAIN-LIKE PROTEINASE OF MHV-A59 - IDENTIFICATION OF A NEW IN-VITRO CLEAVAGE SITE, Virology, 209(2), 1995, pp. 489-497
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.