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 pp lab, is linked to a series of proteolytic events involving virus encod ed proteinases. In this study, we used transfection and immunoprecipit ation assays to show that the human coronavirus 229E-encoded papain-li ke cysteine proteinase PCP1, is responsible for the release of an amin o-terminal protein, p9, from the gene 1-encoded polyproteins. The same protein, p9, has also been identified in virus-infected cells, Furthe rmore. using an in vitro trans-cleavage assay, we defined the proteoly tic cleavage site at the carboxyl terminus of p9 as ppla-pplab amino a cids Gly-111 and Asn-112. These results and a comparative sequence ana lysis 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 deletio n mutagenesis, we were also able to locate the boundaries of the activ e PCP1 domain between ppla-pplab amino acids Gly-861-Glu-975 and Asn-1 209-Gln-1285. Finally. codon mutagenesis nas used to show that Cys-105 4 and His-1205 are essential for PCP1 proteolytic activity suggesting that these amino acids most likely have a catalytic function.