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

作者：Gottipati Keerthi; Acholi Sudheer; Ruggli Nicolas; Choi Kyung H^*

来源：Virology, 2014, 452: 303-309.

DOI：10.1016/j.virol.2014.01.026

• Pestivirus
• Classical swine fever virus
• N-terminal protease N-pro
• Cysteine protease
• Autoprotease
• Self-cleavage
• Substrate specificity
• Mass spectrometry

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%26apos;s interferon response by inducing degradation of interferon regulatory factor-3. N(pro%26apos;)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%26apos;)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.

• 出版日期2014-3