An integrated proteomics approach identifies phosphorylation sites on viral and host proteins that regulate West Nile virus infection.

Upon infection, viruses alter the proteome, creating a hospitable environment for infection. Cells respond to limit viral replication, including through protein regulation by post-translational modifications. We use mass spectrometry to define proteome alterations during West Nile virus (WNV) infection. Our studies identify upregulation of HERPUD1, which restricts WNV replication through a mechanism independent of its role in endoplasmic reticulum (ER)-associated degradation (ERAD). We also identify modifications on viral proteins, including a WNV NS3 phosphorylation site that impacts viral replication. Finally, we reveal activation of two host kinases with antiviral activity. We identify phosphorylation at S108 of AMPKβ1, a non-catalytic subunit that regulates activity of the AMPK complex. We also show activation of PAK2 by phosphorylation at S141, which restricts translation of the viral genome. This work contributes to our understanding of the interplay between host and virus while providing a resource to define the changes to the proteome that regulate viral infection.