Further requirements for cleavage by the murine coronavirus 3C-like proteinase: identification of a cleavage site within ORF1b.
The coronavirus mouse hepatitis virus strain A59 (MHV-A59) encodes a 3C-like proteinase (3CLpro) that is proposed to be responsible for the majority of the processing events that take place within the replicase polyproteins pp1a and pp1ab. In this study we demonstrate that the Q939/S940 peptide bond, located between the polymerase and Zn-finger regions of pp1ab (the POL/Zn site), is processed by the 3CLpro, albeit inefficiently. Mutagenesis of the POL/Zn site, as well as the previously identified HD1/3C site in the 1a region of pp1a and pp1ab, demonstrated that the amino acid residues at the P2 and P1 positions of the cleavage site, occupied by L and Q, respectively, were important determinants of 3CLpro substrate specificity. Finally, a direct comparison of the 3CLpro-mediated cleavages at the HD1/3C and POL/Zn sites was made by determining the rate constants using synthetic peptides. The results show that while a larger polypeptide substrate carrying the HD1/3C site was processed more efficiently than a polypeptide substrate carrying the POL/Zn site, cleavage of the synthetic peptide substrates containing these two cleavage sites occurred at similar efficiencies. This indicates that the overall conformation of a large polyprotein substrate is important in the accessibility of the cleavage site to the proteinase.
Piñón
J D
JD
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, USA.
Teng
H
H
Weiss
S R
SR
D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence / Q000235:genetics; D017934:Coronavirus / Q000201:enzymology* / Q000235:genetics; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
D010455:Peptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases