Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity.
The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large 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, expressed 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 one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain. Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide 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 cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E.
Ziebuhr
J
J
Institute of Virology, University of Würzburg, Germany.
Herold
J
J
Siddell
S G
SG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017934:Coronavirus / Q000201:enzymology*; D028941:Coronavirus 229E, Human; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014764:Viral Proteins / Q000502:physiology*
D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases