Characterization of the leader papain-like protease of MHV-A59.
Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) genome predicts the presence of two papain-like proteases encoded within the first open reading frame of the replicase gene. The more 5' of these domains, the leader papain-like protease, is responsible for the cleavage of the amino terminal protein, p28. We have defined the core of this protease to between amino acids 1075 and 1344 from the beginning of ORF 1a. Deletion analysis coupled with in vitro expression, was used to study p28 cleavage by this leader protease. Expression of a series of deletion mutants showed processing of p28, albeit at lower levels in some of them. Reduced p28 production resulting from a 0.4 kb deletion positioned between p28 and the protease domain suggests an involvement of this region in catalytic processing. Some mutants display cleavage patterns indicative of a second cleavage site. Interestingly, this newly identified cleavage site maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59 infected cells. Mutagenesis of the catalytic H1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like protease encoded in ORF 1a.
Bonilla
P J
PJ
Department of Microbiology, University of Pennsylvania, Philadelphia 19104-6076, USA.
Piñón
J L
JL
Hughes
S
S
Weiss
S R
SR
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis*; D017931:DNA Primers; D004926:Escherichia coli; D005814:Genes, Viral; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010206:Papain; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014764:Viral Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D014764:Viral Proteins; C087637:gene 1 protein, Coronavirus; D012324:RNA-Dependent RNA Polymerase; D003546:Cysteine Endopeptidases; D010206:Papain