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Title: Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

Pestivirus N{sup 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{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup 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{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup 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{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168more » at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.« less
Authors:
;  [1] ;  [2] ;  [1]
  1. Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)
  2. Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland)
Publication Date:
OSTI Identifier:
22435021
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 452-453; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CARBON; CATALYSIS; CLEAVAGE; CYSTEINE; FEVER; HOST; IN VITRO; INTERFERON; LOSSES; MASS SPECTROSCOPY; POLYPEPTIDES; RESIDUES; SALTS; SPECIFICITY; STABILITY; SUBSTRATES; SWINE; VIRUSES