Hydrolysis of a series of synthetic peptide substrates by the human rhinovirus 14 3C proteinase, cloned and expressed in Escherichia coli.
The 3C proteins of several picornaviruses, including poliovirus, foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV), have been demonstrated to be cysteine-type proteinases, involved in the processing of the respective polyproteins expressed by the monocistronic RNA genome. Nucleotide sequencing data have indicated that the human rhinovirus 14 (HRV-14) RNA genome encodes a homologous 3C protein. The HRV-14 3C protein was purified to homogeneity from Escherichia coli expressing the cloned 3C genomic fragment. The enzyme was assayed against peptides corresponding to those residues, predicted (by nucleotide sequencing data) to occur at authentic cleavage sites within the polyprotein. The peptides representing the 1B/1C, 2A/2B, 2C/3A, 3A/3B, 3B/3C and 3C/3D cleavage sites, where proteolysis was predicted to occur at a Gln-Gly junction, were all cleaved by the 3C proteinase. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase. By contrast, peptides corresponding to the predicted 2B/2C and 1C/1D cleavage sites, where the processing was presumed to occur at a Gln-Ala or Glu-Gly bond respectively, were not cleaved by the 3C proteinase. The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys----Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV-14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile. The HRV-14 3C proteinase probably plays an important role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy.
Orr
D C
DC
Department of Virology, Glaxo Group Research Limited, Greenford, Middlesex, U.K.
Long
A C
AC
Kay
J
J
Dunn
B M
BM
Cameron
J M
JM
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D002413:Cations, Divalent; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics; D011994:Recombinant Proteins; D012229:Rhinovirus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature; D014764:Viral Proteins
D002413:Cations, Divalent; D011480:Protease Inhibitors; D011506:Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases