Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N^pro

Pestivirus N^pro is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N^pro blocks the host[U+05F3]s interferon response by inducing degradation of interferon regulatory factor-3. N^pro[U+05F3]s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N^pro-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N^pro proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N^pro[U+05F3]s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N^pro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus.