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 1-encoded polyproteins, pp1a and pp1ab, 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 pp1a-pp1ab 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 pp1a-pp1ab amino acids Gly-861-Glu-975 and Asn-1209-Gln-1285. Finally, codon mutagenesis was 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.
Herold
J
J
Institute of Virology, University of Würzburg, Germany. viro008@mail.uni-wuerzburg.de
Gorbalenya
A E
AE
Thiel
V
V
Schelle
B
B
Siddell
S G
SG
D000595:Amino Acid Sequence; D017934:Coronavirus / Q000235:genetics*; D028941:Coronavirus 229E, Human; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010206:Papain / Q000235:genetics* / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D011506:Proteins; D014764:Viral Proteins; D010206:Papain