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. 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
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.