Protein Kinase G Phosphorylates Mosquito-Borne Flavivirus NS5

Serine/threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to compare the phosphorylation sites of the NS5 proteins of yellow fever virus (YFV) and dengue virus (DENV), two flaviviruses transmitted by mosquitoes. Seven DENV phosphopeptides were identified, but only one conserved phosphoacceptor site (threonine 449 in DENV) was identified in both viruses. This site is predicted to be a protein kinase G (PKG) recognition site and is a strictly conserved serine/threonine phosphoacceptor site in mosquito-borne flaviviruses. In contrast, in tick-borne flaviviruses, this residue is typically a histidine. A DENV replicon engineered to have the tick-specific histidine residue at this position is replication defective. We show that DENV NS5 purified from Escherichia coli is a substrate for PKG in vitro and facilitates the autophosphorylation of PKG as seen with cellular substrates. Phosphorylation in vitro by PKG also occurs at threonine 449. Activators and inhibitors of PKG modulate DENV replication in cell culture but not replication of the tick-borne langat virus. Collectively, these data argue that PKG mediates a conserved serine/threonine phosphorylation event specifically for flaviviruses spread by mosquitoes.The flavivirus genus contains many medically important species, including dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV). More than 2 billion people are at risk of infection by DENV alone, leading to an estimated 50 million cases annually, which may increase further as the range of the mosquito vector expands with urbanization (24). While disease from mosquito-borne flaviviruses is particularly common, there are other flaviviral human pathogens that exist with transmission cycles that do not involve mosquitoes. Tick-borne transmission is the other well-described route, but non-arthropod-borne routes also exist (for example, bats). It is likely that each transmission route has genetic adaptations that facilitate that route, but such changes are not yet understood (7).Serine/threonine phosphorylation is a conserved feature across all three genera of the family Flaviviridae, including the genus flavivirus (the others genera being pestivirus and hepacivirus). Among the features of Flaviviridae, the most-studied examples are the multiple phosphorylations of nonstructural protein 5A (NS5A) of hepatitis C virus, which exists in both basal (termed p56) and hyperphosphorylated (termed p58) states mediated by multiple kinases that both are necessary for and limit replication (14, 18, 23). Phosphorylation of NS5B, the RNA-dependent RNA polymerase (RdRP), has also been shown to affect replicon activity (10). In the genus flavivirus, several mosquito-borne viruses (DENV, WNV, and YFV) and at least one tick-borne encephalitis virus are known to have phosphorylated forms of nonstructural protein NS5 (2, 9, 11, 13, 19). In the genus flavivirus, NS5 is central to viral replication, as it possesses both RdRP and methyltransferase activities. DENV phosphorylation of NS5 correlates with the loss of NS5 interactions with the viral helicase NS3. A hyperphosphorylated form of NS5 was found to localize to the nucleus, away from the cytoplasmic sites of viral replication (6, 9). A nuclear localization sequence is present in DENV NS5 and is phosphorylated in vitro by host CKII, but the relationship between phosphorylation and nuclear localization has yet to be fully elucidated (17). Multiple different serine/threonine phosphorylation events likely occur in the flaviviral life cycle, potentially affecting various functions of NS5 (2), but the role of these events and identity of the kinase(s) responsible are largely unknown.In this report, we used mass spectrometry to identify serine/threonine phosphorylation sites in DENV. A single phosphoacceptor site, previously identified in YFV, is conserved specifically in the mosquito-borne flaviviruses but not the tick-borne flaviviruses. Furthermore, in vitro studies reveal that this site is phosphorylated by a cyclic-nucleotide-dependent kinase, protein kinase G (PKG), and a phosphoacceptor threonine/serine is required for replication. Taken together, these data implicate the PKG pathway in flaviviral replication for the first time and suggest a host cell pathway that could be targeted by antiviral therapy.