Biologically active protease 3C of human rhinovirus 1A is expressed from a cloned cDNA segment in Escherichia coli.
We produced the putative protease 3C of human rhinovirus 1A (HRV-1A), a minor group rhinovirus, by Escherichia coli expression of a segment of HRV-1A cDNA coding for 3A, 3B, 3C, and parts of 2C and 3D (delta 2C3ABC delta 3D). The protease 3C was expected to be processed by intramolecular Q-G cleavages from the virus-specific precursor polypeptide. While the N-terminal 3B-3C site was correctly cleaved, the C-terminal Q-G site of 3C was not processed. Western blotting with a site-specific polyclonal antipeptide antibody showed that not the mature 3C polypeptide, but the 3C-containing precursor 3C delta 3D was the only rhinovirus-specific protein. Mature 3C was obtained by introducing two stop codons at positions glycine-1 and glutamine-2 of 3D by site-specific mutagenesis. This mutant produced the mature 3C protease of HRV-1A. In contrast to poliovirus, the mature 3C protease of HRV-1A is a minor peptide in virus-infected HeLa cells. The 3C protein can be detected only by Western blotting with a polyclonal antipeptide 3C antibody but not by radiolabeling the viral polypeptide.
Aschauer
B
B
Sandoz Forschungsinstitut, Vienna, Austria.
Werner
G
G
McCray
J
J
Rosenwirth
B
B
Bachmayer
H
H
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D004252:DNA Mutational Analysis; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012229:Rhinovirus / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics
D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases