Zinc binding in pestivirus N(pro) is required for interferon regulatory factor 3 interaction and degradation.
Pestiviruses, such as bovine viral diarrhea virus and classical swine fever virus (CSFV), use the viral protein N(pro) to subvert host cell antiviral responses. N(pro) 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. N(pro) 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 N(pro). We determined that N(pro) contains a novel metal-binding TRASH motif consisting of Cys-X(21)-Cys-X(3)-Cys (where X is any amino acid) at its C-terminus. We also found that N(pro) 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 N(pro) abolished the interaction of N(pro) with IRF3 and resulted in the loss of virus-mediated IRF3 degradation in CSFV-infected cells. Thus, the zinc-binding ability of N(pro) in pestiviruses appears to be essential for the virus-mediated degradation of IRF3.
Szymanski
Michal R
MR
Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch at Galveston, TX 77555-0647, USA.
Fiebach
Ana R
AR
Tratschin
Jon-Duri
JD
Gut
Marco
M
Ramanujam
V M Sadagopa
VM
Gottipati
Keerthi
K
Patel
Purvi
P
Ye
Mengyi
M
Ruggli
Nicolas
N
Choi
Kyung H
KH
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001908:Diarrhea Viruses, Bovine Viral / Q000378:metabolism*; D017930:Genes, Reporter; D050838:Interferon Regulatory Factor-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D050838:Interferon Regulatory Factor-3; D008667:Metalloproteins; D019590:Nucleocapsid Proteins; D001224:Aspartic Acid; D015032:Zinc; D003545:Cysteine