Development of in vitro peptide substrates for human rhinovirus-14 2A protease.
Purified 2A protease from human rhinovirus serotype-14 (HRV14) was unable to efficiently cleave a 16-mer peptide representing its authentic cis-cleavage site on the viral polyprotein, implying that in vivo cis cleavage by this enzyme might be very different from its in vitro trans activity. Presence of a serine at position P2 and a leucine at P2' in the 16-mer peptide was found to be responsible for the low peptide cleavage efficiency. To search for an efficient peptide substrate for HRV14 2A, small peptides derived from other rhinovirus 2A protease cleavage sites were synthesized and tested. These results suggested that the N-terminal 8 amino acids were sufficient for HRV14 2A cleavage to occur, although the P1' and P2' residue identities were important to the cleavage of peptides with amino acids occupying both sides of the scissile bond. On the basis of the 2A substrate requirements, a sensitive fluorometric assay for the viral 2A proteases was developed using peptides with anthranilide and 3-nitrotyrosine as the resonance energy transfer donor/quencher pair. Our data indicated that these fluorescent peptide substrates were suitable for 2A protease characterization and inhibitor evaluation.
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, 46285, USA. qmwang@lilly.com
Q M
QM
Wang
R B
RB
Johnson
W
W
Sommergruber
T A
TA
Shepherd
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D012229:Rhinovirus / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014764:Viral Proteins
D005456:Fluorescent Dyes; D009842:Oligopeptides; D011480:Protease Inhibitors; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus