Proteolytic processing at the amino terminus of human coronavirus 229E gene 1-encoded polyproteins: Identification of a papain-like proteinase and its substrate

Expression of the coronavirus gene I-encoded polyproteins. ppla and pplab, is linked to a series of proteolytic events involving virus encoded proteinases. In this study, we used transfection and immunoprecipitation assays to show that the human coronavirus 229E-encoded papain-like cysteine proteinase PCP1, is responsible for the release of an amino-terminal protein, p9, from the gene 1-encoded polyproteins. The same protein, p9, has also been identified in virus-infected cells, Furthermore. using an in vitro trans-cleavage assay, we defined the proteolytic cleavage site at the carboxyl terminus of p9 as ppla-pplab amino acids Gly-111 and Asn-112. These results and a comparative sequence analysis suggest that substrate positions P1 and P5 seem to be the major determinants of the PCP1 cleavage site and that the latter can occupy a variable position at the amino terminus of the coronavirus ppla and pplab polyproteins. By combining the trans-cleavage assay with deletion mutagenesis, we were also able to locate the boundaries of the active PCP1 domain between ppla-pplab amino acids Gly-861-Glu-975 and Asn-1209-Gln-1285. Finally. codon mutagenesis nas used to show that Cys-1054 and His-1205 are essential for PCP1 proteolytic activity suggesting that these amino acids most likely have a catalytic function.