title: Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro.
abstract: 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׳s interferon response by inducing degradation of interferon regulatory factor-3. N(pro׳)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׳)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.
authors.affiliation: Department of Biochemistry & Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647, USA.
authors.forename: Keerthi
authors.initials: K
authors.lastname: Gottipati
authors.affiliation: Department of Biochemistry & Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647, USA.
authors.forename: Sudheer
authors.initials: S
authors.lastname: Acholi
authors.affiliation: Institute of Virology and Immunology, CH-3147 Mittelhäusern, Switzerland.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Ruggli
authors.affiliation: Department of Biochemistry & Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647, USA. Electronic address: kychoi@utmb.edu.
authors.forename: Kyung H
authors.initials: KH
authors.lastname: Choi
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010576:Pestivirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D012697:Serine Endopeptidases; C036777:protease N
keywords: Autoprotease; Classical swine fever virus; Cysteine protease; Mass spectrometry; N-terminal protease N(pro); Pestivirus; Self-cleavage; Substrate specificity