3C-LIKE PROTEASE OF RABBIT HEMORRHAGIC-DISEASE VIRUS - IDENTIFICATIONOF CLEAVAGE SITES IN THE ORF1 POLYPROTEIN AND ANALYSIS OF CLEAVAGE SPECIFICITY

Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as par t of a large polyprotein. Upon expression in Escherichia coli, the pro tease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-termi nal sequence analysis of the cleavage products, Cleavage at the N term inus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid s ubstitutions were introduced at the P2, P1, and P1' positions at the c leavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important d eterminant for substrate recognition. Only glutamic acid, glutamine, a nd aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the pro tease, but a number of amino acids with larger side chains were also t olerated. Substitutions at the P2 position had only little effect on t he cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at t he junction of the RNA polymerase and the capsid protein, An EG dipept ide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a t rypsin-like cysteine protease that is similar to 3C proteases with reg ard to function and specificity but is more similar to 2A proteases wi th regard to size.