CHARACTERIZATION OF A HUMAN CORONAVIRUS (STRAIN 229E) 3C-LIKE PROTEINASE ACTIVITY

The RNA polymerase gene of human coronavirus (HCV) 229E encodes a larg e polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expre ssed the putative HCV 229E 3C-like proteinase domain as part of a beta -galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of on e amino acid within the predicted proteinase domain (His-3006-->Asp-30 06) abolishes, or at least significantly reduces, this activity. Amino -terminal sequence analysis of a purified, 34-kDa cleavage product sho ws that the bacterial fusion protein is cleaved at the dipeptide Gln-2 965-Ala-2966, which is the predicted amino-terminal end of the putativ e 3C-like proteinase domain, Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid s equence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading fra me 1b of the RNA polymerase gene. Finally, using fusion protein-specif ic antiserum, we have identified a 34-kDa, 3C-like proteinase polypept ide in HCV 229E-infected MRC-5 cells, This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cel l in very low amounts. These data contribute to the characterization o f the 3C-like proteinase activity of HCV 229E.