Quantitative Label-Free Phosphoproteomics Reveals Differentially Regulated Protein Phosphorylation Involved in West Nile Virus-Induced Host Inflammatory Response.
West Nile virus (WNV) can cause neuro-invasive and febrile illness that may be fatal to humans. The production of inflammatory cytokines is key to mediating WNV-induced immunopathology in the central nervous system. Elucidating the host factors utilized by WNV for productive infection would provide valuable insights into the evasion strategies used by this virus. Although attempts have been made to determine these host factors, proteomic data depicting WNV-host protein interactions are limited. We applied liquid chromatography-tandem mass spectrometry for label-free, quantitative phosphoproteomics to systematically investigate the global phosphorylation events induced by WNV infection. Quantifiable changes to 1,657 phosphoproteins were found; of these, 626 were significantly upregulated and 227 were downregulated at 12 h postinfection. The phosphoproteomic data were subjected to gene ontology enrichment analysis, which returned the inflammation-related spliceosome, ErbB, mitogen-activated protein kinase, nuclear factor kappa B, and mechanistic target of rapamycin signaling pathways. We used short interfering RNAs to decrease the levels of glycogen synthase kinase-3 beta, bifunctional polynucleotide phosphatase/kinase, and retinoblastoma 1 and found that the activity of nuclear factor kappa B (p65) is significantly decreased in WNV-infected U251 cells, which in turn led to markedly reduced inflammatory cytokine production. Our results provide a better understanding of the host response to WNV infection and highlight multiple targets for the development of antiviral and anti-inflammatory therapies.
Hao
H
Zhang
Jun
J
Sun
Jing
J
Ye
Usama
U
Ashraf
Zheng
Z
Chen
Bibo
B
Zhu
Wen
W
He
Qiuping
Q
Xu
Yanming
Y
Wei
Huanchun
H
Chen
Department of Pathology, University of Georgia , Athens, Georgia 30602, United States.
Zhen F
ZF
Fu
Rong
R
Liu
Shengbo
S
Cao
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D016207:Cytokines / Q000096:biosynthesis; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D063990:Gene Ontology; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D054884:Host-Pathogen Interactions; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D060066:Protein Interaction Maps; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D014901:West Nile Fever / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014902:West Nile virus / Q000472:pathogenicity*
D016207:Cytokines; D018836:Inflammation Mediators; D016328:NF-kappa B; D010750:Phosphoproteins; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D010770:Phosphotransferases; C530380:PNKP protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D045643:DNA Repair Enzymes
West Nile virus; inflammatory response; liquid chromatography-tandem mass spectrometry; phosphorylation