Abstract
BackgroundThe 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.MethodsWe 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.ResultsWe 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.ConclusionsPKG 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.
Highlights
The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown
West Nile virus (WNV) NS5 is phosphorylated in infected HEK293T cells To identify specific sites that are phosphorylated in WNV NS5 during an infection, HEK293T cells were infected with WNV at a multiplicity of infection (MOI) of 10, and cell lysates were harvested at 24 hours postinfection
The data here suggest further substrate specificities for mammalian Protein kinase G (PKG) between mosquito- and tick-borne flaviviral NS5, as LGTV NS5 does not interact with PKG as WNV NS5 does
Summary
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. A number of cellular kinases are able to phosphorylate viral proteins from a wide range of viruses, and the roles of kinases in viral lifecycles continue to be studied [11]. The identity of many kinases able to phosphorylate flaviviral NS5, and the role of these kinases in viral infections, are unknown. We have previously demonstrated mammalian protein kinase G (PKG), a cGMP-dependent serine/threonine kinase, is able to phosphorylate dengue (DENV-2) NS5 at a site conserved among all mosquitoborne flaviviruses (Thr449, in the RdRp domain of NS5) [15]. DENV-2 NS5 is phosphorylated by mosquito PKG, and alterations in PKG activity changes the flight behavior of Aedes and non-Aedes mosquitoes [16] raising the possibility that flaviviruses alter their own transmission from vectors
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