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.