Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site.
Coronavirus infectious bronchitis virus (IBV) encodes a trypsin-like proteinase (3C-like proteinase) by ORF 1a, which has been demonstrated to play a pivotal role in proteolytic processing of gene 1-encoded polyproteins. In our previous studies, the proteinase was identified as a 33-kDa protein in IBV-infected cells, and its catalytic center was shown to consist of H(2820) and C(2922) residues. It is released from the 1a and 1a/1b polyproteins by autoprocessing at two Q-S dipeptide bonds (Q(2779)-S(2780) and Q(3086)-S(3087)). In this report, further characterization of the two cleavage sites demonstrates that the N-terminal Q(2779)-S(2780) site is tolerant to mutations at the P1 position. Deletion of the C-terminal region of the proteinase shows that a significant amount of the enzymatic activity is maintained upon deletion of up to 67 amino acids, suggesting that the extreme C-terminal region may be dispensable for the proteolytic activity of the proteinase. Analysis of the autoprocessing kinetics in vitro reveals that proteolysis at the Q(2779)-S(2780) site is the first cleavage event mediated by this proteinase. This is followed by cleavage at the Q(3086)-S(3087) site. The occurrence of both cleavage events in intact cells is potentially rapid and efficient, as no intermediate cleavage products covering the proteinase were detected in either IBV-infected or transfected cells. Immunofluorescence microscopy and subcellular fractionation studies further show differential subcellular localization of the proteinase in IBV-infected cells and in cells expressing the 3C-like proteinase alone, indicating that additional roles in viral replication might be played by this protein. Finally, a Q-A (Q(3379)-A(3380)) dipeptide bond encoded by nucleotides 10,663 to 10,668 was demonstrated to be a cleavage site of the proteinase.
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L F
LF
Institute of Molecular Agrobiology, The National University of Singapore, 1 Research Link, 117604, Singapore.
Liu
D X
DX
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D001351:Infectious bronchitis virus / Q000201:enzymology* / Q000235:genetics; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D014162:Transfection; D014709:Vero Cells
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases; C000706208:3C-like proteinase, Avian infectious bronchitis virus; D000086782:Coronavirus 3C Proteases