DEVELOPMENT OF IN-VITRO PEPTIDE-SUBSTRATES FOR HUMAN RHINOVIRUS-14 2APROTEASE

Purified 2A protease from human rhinovirus serotype-14 (HRV14) was una ble 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 tran s activity. Presence of a serine at position P2 and a leucine at P2' i n the 16-mer peptide was found to be responsible for the low peptide c leavage efficiency. To search for an efficient peptide substrate for H RV14 2A, small peptides derived from other rhinovirus 2A protease clea vage sites were synthesized and tested. These results suggested that t he N-terminal 8 amino acids were sufficient for HRV14 2A cleavage to o ccur, although the P1' and P2' residue identities were important to th e cleavage of peptides with amino acids occupying both sides of the sc issile bond. On the basis of the 2A substrate requirements, a sensitiv e fluorometric assay for the viral 2A proteases was developed using pe ptides with anthranilide and 3-nitrotyrosine as the resonance energy t ransfer donor/quencher pair. Our data indicated that these fluorescent peptide substrates were suitable for 2A protease characterization and inhibitor evaluation. (C) 1998 Academic Press.