title: West Nile virus methyltransferase domain interacts with protein kinase G.
abstract: BACKGROUND
  The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5.
  
  
  METHODS
  We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering.
  
  
  RESULTS
  We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication.
  
  
  CONCLUSIONS
  PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication.
authors.affiliation: Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Keating
authors.forename: Dipankar
authors.initials: D
authors.lastname: Bhattacharya
authors.forename: Pei-Yin
authors.initials: PY
authors.lastname: Lim
authors.forename: Shaun
authors.initials: S
authors.lastname: Falk
authors.forename: Bernard
authors.initials: B
authors.lastname: Weisblum
authors.forename: Kristen A
authors.initials: KA
authors.lastname: Bernard
authors.forename: Mayuri
authors.initials: M
authors.lastname: Sharma
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Kuhn
authors.forename: Rob
authors.initials: R
authors.lastname: Striker
mesh_terms: D000818:Animals; D002460:Cell Line; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004252:DNA Mutational Analysis; D054884:Host-Pathogen Interactions; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014902:West Nile virus / Q000502:physiology*
chemical_list: D050505:Mutant Proteins; C096041:NS5 protein, flavivirus; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; D017869:Cyclic GMP-Dependent Protein Kinases