Expression and purification of recombinant rhinovirus 14 3CD proteinase and its comparison to the 3C proteinase.
Human rhinovirus (HRV) is a positive-stranded RNA virus with an open reading frame that encodes for a single polyprotein of about 3000 amino acids. The HRV polyprotein is proteolytically processed; eight of nine cleavages are catalyzed by the 3C and/or the 3CD proteinases. We have expressed and purified recombinant HRV14 3C and 3CD proteinases and investigated their substrate selectivity and inhibitor sensitivity. Expressed 3CD proteinase had the P1/P1' residues of the 3C/3D cleavage site mutated from Gln/Gly to Ala/Ala in order to prevent autocleavage. The 3CD proteinase activities were measured by utilization of native, chromogenic, and fluorogenic peptide substrates. The 3CD proteinase exhibited < or =15% activity, compared to 3C, toward peptidyl p-nitroanilide substrates which contain only the p-nitroaniline moiety in the prime side. The 3C and 3CD proteinases exhibited similar activities for both internally quenched fluorogenic and native peptides. These results suggest that the two enzymes have similar but slightly different substrate specificity, especially on their preference for prime side residues. Inhibitor sensitivities toward classical proteinase inhibitors were generally similar for both enzymes. Small peptidyl inhibitors, specifically designed and synthesized for HRV14 3C, also inhibited the 3CD proteinase. Taken together, our data indicate that the 3D domain of 3CD proteinase had some influence on substrate recognition, but did not have dramatic impact on its interaction with inhibitors.
Davis
G J
GJ
Infectious Diseases Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Wang
Q M
QM
Cox
G A
GA
Johnson
R B
RB
Wakulchik
M
M
Dotson
C A
CA
Villarreal
E C
EC
D000086042:3C Viral Proteases; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D012229:Rhinovirus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014764:Viral Proteins
D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases