Processing of the human coronavirus 229E replicase polyproteins by the virus-encoded 3C-like proteinase: Identification of proteolytic products and cleavage sites common to pp1a and pp1ab

Citation
J. Ziebuhr et Sg. Siddell, Processing of the human coronavirus 229E replicase polyproteins by the virus-encoded 3C-like proteinase: Identification of proteolytic products and cleavage sites common to pp1a and pp1ab, J VIROLOGY, 73(1), 1999, pp. 177-185
Citations number
45
Categorie Soggetti
Microbiology
Journal title
JOURNAL OF VIROLOGY
ISSN journal
0022538X → ACNP
Volume
73
Issue
1
Year of publication
1999
Pages
177 - 185
Database
ISI
SICI code
0022-538X(199901)73:1<177:POTHC2>2.0.ZU;2-#
Abstract
Replicase gene expression by the human coronavirus 229E involves the synthe sis of two large polyproteins, pp1a and pp1ab. Experimental evidence sugges ts that these precursor molecules are subject to extensive proteolytic proc essing. In this study, we show that a chymotrypsin-like enzyme, the virus-e ncoded 3C-like proteinase (3CL(pro)), cleaves within a common region of pp1 a and pp1ab (amino acids 3490 to 4068) at four sites. trans-cleavage assays revealed that polypeptides of 5, 23, 12, and 16 kDa are processed from pp1 a/pp1ab by proteolysis of the peptide bonds Q3546/S3547, Q3629/S3630, Q3824 /N3825, and Q3933/A3934. Relative rate constants for the 3CL(pro)-mediated cleavages Q2965/A2966, Q3267/S3268, Q3824/N3825, and Q3933/A3934 were deriv ed by competition experiments using synthetic peptides and recombinant 3CL( pro). The results indicate that coronavirus cleavage sites differ significa ntly with regard to their susceptibilities to proteolysis by 3CL(pro), Fina lly, immunoprecipitation with specific rabbit antisera was used to detect t he pp1a/pp1ab processing end products in virus-infected cells, and immunofl uorescence data that suggest an association of these polypeptides with intr acellular membranes were obtained.