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

Keerthi Gottipati, Sudheer Acholi, Nicolas Ruggli, Kyung Choi

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Pestivirus Npro 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, Npro blocks the host[U+05F3]s interferon response by inducing degradation of interferon regulatory factor-3. Npro[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 Npro-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of Npro 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 Npro[U+05F3]s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, Npro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus.

Original languageEnglish (US)
Pages (from-to)303-309
Number of pages7
JournalVirology
Volume452-453
DOIs
StatePublished - 2014

Fingerprint

Pestivirus
Substrate Specificity
Peptide Hydrolases
Proteins
Interferon Regulatory Factor-3
Protein Stability
Interferons
Catalytic Domain
Salts
Peptides

Keywords

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

ASJC Scopus subject areas

  • Virology

Cite this

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro . / Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung.

In: Virology, Vol. 452-453, 2014, p. 303-309.

Research output: Contribution to journalArticle

Gottipati, Keerthi ; Acholi, Sudheer ; Ruggli, Nicolas ; Choi, Kyung. / Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro In: Virology. 2014 ; Vol. 452-453. pp. 303-309.
@article{2aaabc4e6ce74cf6b77fac5aecabc54f,
title = "Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro",
abstract = "Pestivirus Npro 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, Npro blocks the host[U+05F3]s interferon response by inducing degradation of interferon regulatory factor-3. Npro[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 Npro-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of Npro 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 Npro[U+05F3]s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, Npro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus.",
keywords = "Autoprotease, Classical swine fever virus, Cysteine protease, Mass spectrometry, Pestivirus, Self-cleavage, Substrate specificity",
author = "Keerthi Gottipati and Sudheer Acholi and Nicolas Ruggli and Kyung Choi",
year = "2014",
doi = "10.1016/j.virol.2014.01.026",
language = "English (US)",
volume = "452-453",
pages = "303--309",
journal = "Virology",
issn = "0042-6822",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro

AU - Gottipati, Keerthi

AU - Acholi, Sudheer

AU - Ruggli, Nicolas

AU - Choi, Kyung

PY - 2014

Y1 - 2014

N2 - Pestivirus Npro 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, Npro blocks the host[U+05F3]s interferon response by inducing degradation of interferon regulatory factor-3. Npro[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 Npro-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of Npro 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 Npro[U+05F3]s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, Npro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus.

AB - Pestivirus Npro 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, Npro blocks the host[U+05F3]s interferon response by inducing degradation of interferon regulatory factor-3. Npro[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 Npro-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of Npro 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 Npro[U+05F3]s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, Npro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus.

KW - Autoprotease

KW - Classical swine fever virus

KW - Cysteine protease

KW - Mass spectrometry

KW - Pestivirus

KW - Self-cleavage

KW - Substrate specificity

UR - http://www.scopus.com/inward/record.url?scp=84896278871&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84896278871&partnerID=8YFLogxK

U2 - 10.1016/j.virol.2014.01.026

DO - 10.1016/j.virol.2014.01.026

M3 - Article

VL - 452-453

SP - 303

EP - 309

JO - Virology

JF - Virology

SN - 0042-6822

ER -