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) genome predicts the presence of two papain-like proteinases encoded within the first open reading frame (ORF 1a) of the replicase gene. The more 5' of these domains, the leader papain-like proteinase, is responsible for the cleavage of the amino terminal protein, p28. The core of this proteinase domain was defined to between amino acids 1084 and 1316 from the beginning of ORF 1a. Through the use of deletion analysis coupled with in vitro expression, we studied the role of the coding region between p28 and the leader papain-like proteinase on the cleavage of p28 itself. Expression of a series of deletion mutants showed processing of p28, albeit at lower levels. Reduced p28 production resulting from a 0.4-kb deletion positioned between p28 and the proteinase domain suggests an involvement of this region in catalytic processing. Some mutants displayed cleavage patterns indicative of a second cleavage site. Interestingly, this new cleavage site identified in vitro maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59-infected cells. Mutagenesis of the catalytic His1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like proteinase encoded in ORF 1a.
Bonilla
P J
PJ
Department of Microbiology, University of Pennsylvania, Philadelphia 19104-6076, USA.
Hughes
S A
SA
Piñón
J D
JD
Weiss
S R
SR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D000086882:Coronavirus Papain-Like Proteases; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D016679:Genome, Viral; D006639:Histidine; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010206:Papain / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D016133:Polymerase Chain Reaction; D016364:Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D017384:Sequence Deletion
D017931:DNA Primers; D000939:Epitopes; D011994:Recombinant Proteins; D006639:Histidine; D000086882:Coronavirus Papain-Like Proteases; D010206:Papain