Zinc Binding in Pestivirus Npro Is Required for Interferon Regulatory Factor 3 Interaction and Degradation

Michal R. Szymanski, Ana R. Fiebach, Jon Duri Tratschin, Marco Gut, V-M Ramanujam, Keerthi Gottipati, Purvi Patel, Mengyi Ye, Nicolas Ruggli, Kyung Choi

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Pestiviruses, such as bovine viral diarrhea virus and classical swine fever virus (CSFV), use the viral protein Npro to subvert host cell antiviral responses. Npro is the first protein encoded by the single large open reading frame of the pestivirus positive-sense RNA genome and has an autoproteolytic activity, cleaving itself off from the polyprotein. Npro also targets interferon regulatory factor 3 (IRF3), a transcription factor for alpha/beta interferon genes, and promotes its proteasomal degradation, a process that is independent of the proteolytic activity of Npro. We determined that Npro contains a novel metal-binding TRASH motif consisting of Cys-X21-Cys-X3-Cys (where X is any amino acid) at its C-terminus. We also found that Npro coordinates a single zinc atom as determined by graphite furnace-atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry. Mutational and biochemical analyses show that the cysteine residues in the TRASH motif are required for zinc binding and protein stability. Individual substitutions of the cysteines in the TRASH motif of CSFV Npro abolished the interaction of Npro with IRF3 and resulted in the loss of virus-mediated IRF3 degradation in CSFV-infected cells. Thus, the zinc-binding ability of Npro in pestiviruses appears to be essential for the virus-mediated degradation of IRF3.

Original languageEnglish (US)
Pages (from-to)438-449
Number of pages12
JournalJournal of Molecular Biology
Volume391
Issue number2
DOIs
StatePublished - Aug 14 2009

Fingerprint

Pestivirus
Interferon Regulatory Factor-3
Classical swine fever virus
Zinc
Cysteine
Viruses
Bovine Viral Diarrhea Viruses
Polyproteins
Atomic Spectrophotometry
Graphite
Protein Stability
Interferon-beta
Viral Proteins
Interferon-alpha
Open Reading Frames
Antiviral Agents
Mass Spectrometry
Transcription Factors
Metals
Genome

Keywords

  • interferon regulatory factor 3
  • N-terminal protease N
  • pestivirus
  • proteasomal degradation
  • zinc binding protein

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Zinc Binding in Pestivirus Npro Is Required for Interferon Regulatory Factor 3 Interaction and Degradation. / Szymanski, Michal R.; Fiebach, Ana R.; Tratschin, Jon Duri; Gut, Marco; Ramanujam, V-M; Gottipati, Keerthi; Patel, Purvi; Ye, Mengyi; Ruggli, Nicolas; Choi, Kyung.

In: Journal of Molecular Biology, Vol. 391, No. 2, 14.08.2009, p. 438-449.

Research output: Contribution to journalArticle

Szymanski, MR, Fiebach, AR, Tratschin, JD, Gut, M, Ramanujam, V-M, Gottipati, K, Patel, P, Ye, M, Ruggli, N & Choi, K 2009, 'Zinc Binding in Pestivirus Npro Is Required for Interferon Regulatory Factor 3 Interaction and Degradation', Journal of Molecular Biology, vol. 391, no. 2, pp. 438-449. https://doi.org/10.1016/j.jmb.2009.06.040
Szymanski, Michal R. ; Fiebach, Ana R. ; Tratschin, Jon Duri ; Gut, Marco ; Ramanujam, V-M ; Gottipati, Keerthi ; Patel, Purvi ; Ye, Mengyi ; Ruggli, Nicolas ; Choi, Kyung. / Zinc Binding in Pestivirus Npro Is Required for Interferon Regulatory Factor 3 Interaction and Degradation. In: Journal of Molecular Biology. 2009 ; Vol. 391, No. 2. pp. 438-449.
@article{d97242d2f44c42339c2a3305b7c25e10,
title = "Zinc Binding in Pestivirus Npro Is Required for Interferon Regulatory Factor 3 Interaction and Degradation",
abstract = "Pestiviruses, such as bovine viral diarrhea virus and classical swine fever virus (CSFV), use the viral protein Npro to subvert host cell antiviral responses. Npro is the first protein encoded by the single large open reading frame of the pestivirus positive-sense RNA genome and has an autoproteolytic activity, cleaving itself off from the polyprotein. Npro also targets interferon regulatory factor 3 (IRF3), a transcription factor for alpha/beta interferon genes, and promotes its proteasomal degradation, a process that is independent of the proteolytic activity of Npro. We determined that Npro contains a novel metal-binding TRASH motif consisting of Cys-X21-Cys-X3-Cys (where X is any amino acid) at its C-terminus. We also found that Npro coordinates a single zinc atom as determined by graphite furnace-atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry. Mutational and biochemical analyses show that the cysteine residues in the TRASH motif are required for zinc binding and protein stability. Individual substitutions of the cysteines in the TRASH motif of CSFV Npro abolished the interaction of Npro with IRF3 and resulted in the loss of virus-mediated IRF3 degradation in CSFV-infected cells. Thus, the zinc-binding ability of Npro in pestiviruses appears to be essential for the virus-mediated degradation of IRF3.",
keywords = "interferon regulatory factor 3, N-terminal protease N, pestivirus, proteasomal degradation, zinc binding protein",
author = "Szymanski, {Michal R.} and Fiebach, {Ana R.} and Tratschin, {Jon Duri} and Marco Gut and V-M Ramanujam and Keerthi Gottipati and Purvi Patel and Mengyi Ye and Nicolas Ruggli and Kyung Choi",
year = "2009",
month = "8",
day = "14",
doi = "10.1016/j.jmb.2009.06.040",
language = "English (US)",
volume = "391",
pages = "438--449",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Zinc Binding in Pestivirus Npro Is Required for Interferon Regulatory Factor 3 Interaction and Degradation

AU - Szymanski, Michal R.

AU - Fiebach, Ana R.

AU - Tratschin, Jon Duri

AU - Gut, Marco

AU - Ramanujam, V-M

AU - Gottipati, Keerthi

AU - Patel, Purvi

AU - Ye, Mengyi

AU - Ruggli, Nicolas

AU - Choi, Kyung

PY - 2009/8/14

Y1 - 2009/8/14

N2 - Pestiviruses, such as bovine viral diarrhea virus and classical swine fever virus (CSFV), use the viral protein Npro to subvert host cell antiviral responses. Npro is the first protein encoded by the single large open reading frame of the pestivirus positive-sense RNA genome and has an autoproteolytic activity, cleaving itself off from the polyprotein. Npro also targets interferon regulatory factor 3 (IRF3), a transcription factor for alpha/beta interferon genes, and promotes its proteasomal degradation, a process that is independent of the proteolytic activity of Npro. We determined that Npro contains a novel metal-binding TRASH motif consisting of Cys-X21-Cys-X3-Cys (where X is any amino acid) at its C-terminus. We also found that Npro coordinates a single zinc atom as determined by graphite furnace-atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry. Mutational and biochemical analyses show that the cysteine residues in the TRASH motif are required for zinc binding and protein stability. Individual substitutions of the cysteines in the TRASH motif of CSFV Npro abolished the interaction of Npro with IRF3 and resulted in the loss of virus-mediated IRF3 degradation in CSFV-infected cells. Thus, the zinc-binding ability of Npro in pestiviruses appears to be essential for the virus-mediated degradation of IRF3.

AB - Pestiviruses, such as bovine viral diarrhea virus and classical swine fever virus (CSFV), use the viral protein Npro to subvert host cell antiviral responses. Npro is the first protein encoded by the single large open reading frame of the pestivirus positive-sense RNA genome and has an autoproteolytic activity, cleaving itself off from the polyprotein. Npro also targets interferon regulatory factor 3 (IRF3), a transcription factor for alpha/beta interferon genes, and promotes its proteasomal degradation, a process that is independent of the proteolytic activity of Npro. We determined that Npro contains a novel metal-binding TRASH motif consisting of Cys-X21-Cys-X3-Cys (where X is any amino acid) at its C-terminus. We also found that Npro coordinates a single zinc atom as determined by graphite furnace-atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry. Mutational and biochemical analyses show that the cysteine residues in the TRASH motif are required for zinc binding and protein stability. Individual substitutions of the cysteines in the TRASH motif of CSFV Npro abolished the interaction of Npro with IRF3 and resulted in the loss of virus-mediated IRF3 degradation in CSFV-infected cells. Thus, the zinc-binding ability of Npro in pestiviruses appears to be essential for the virus-mediated degradation of IRF3.

KW - interferon regulatory factor 3

KW - N-terminal protease N

KW - pestivirus

KW - proteasomal degradation

KW - zinc binding protein

UR - http://www.scopus.com/inward/record.url?scp=67650692248&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=67650692248&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2009.06.040

DO - 10.1016/j.jmb.2009.06.040

M3 - Article

VL - 391

SP - 438

EP - 449

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -