The NS5A/NS5 Proteins of Viruses from Three Genera of the Family Flaviviridae Are Phosphorylated by Associated Serine/Threonine Kinases

Abstract

Phosphorylation of the expressed NS5A protein of hepatitis C virus (HCV), a member of the Hepacivirus genus of the familyFlaviviridae, has been demonstrated in mammalian cells and in a cell-free assay by an associated kinase activity. In this report, phosphorylation is also shown for the NS5A and NS5 proteins, respectively, of bovine viral diarrhea virus (BVDV) and yellow fever virus (YF), members of the other two established genera in this family. Phosphorylation of BVDV NS5A and YF NS5 was observed in infected cells, transient expression experiments, and a cell-free assay similar to the one developed for HCV NS5A. Phosphoamino acid analyses indicated that all three proteins were phosphorylated by serine/threonine kinases. Similarities in the properties of BVDV NS5A, YF NS5, and HCV NS5A phosphorylation in vitro further suggested that closely related kinases or the same kinase may phosphorylate these viral proteins. Conservation of this trait among three quite distantly related viruses representing three separate genera suggests that phosphorylation of the NS5A/NS5 proteins or their association with cellular kinases may play an important role in the flavivirus life cycle.

Up-Regulation of Hepatitis C Virus Replication and Production by Inhibition of MEK/ERK Signaling

“…Moreover, selective inhibition of cellular kinases has found an inverse correlation between NS5A phosphorylation level and HCV RNA replication [29]. In addition, NS5A protein is phosphophorylated on serine and threonine residues [30,31] making it a potential substrate for MEK kinase. While one study has shown little change in the level of NS5A phosphorylation after treatment with the MEK kinase inhibitor PD98059 [24], the other one has demonstrated an inverse correlation between inhibition of MEK kinase, the level of NS5A phosphorylation and HCV replication [15].…”

Regulation of positive-strand RNA virus replication: The emerging role of phosphorylation

“…Kim et al (2006) Flaviviridae BVDV NS5A ? Reed et al (1998) Flaviviridae DEN-2 NS5 a Inhibition of interaction between viral replication proteins NS5 and NS3 Kapoor et al (1995) Flaviviridae HCV NS5B a Enhancement of viral RNA synthesis Hwang et al (1997), Kim et al (2004)…”

“…Morozova et al (1997) Flaviviridae YFV NS5 a ? Reed et al (1998) Picornaviridae PV 3D a ? Ransone and Dasgupta (1989) Potyviridae C-terminus, while the 58 kDa protein -referred to as the hyperphosphorylated form -is phosphorylated within a serine-rich central region of the protein (Kaneko et al, 1994;Tanji et al, 1995;Huang et al, 2004).…”

Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis

“…The zinc-binding NS5A proteins of HCV and the pestiviruses 83,84 have essential but undefined roles in RNA replication; no enzymatic activity has been ascribed to them ( Fig 5). For both genera, these proteins are basally phosphorylated on serines by what seems to be the same cellular kinase(s) 85,86 ; HCV NS5A is also a substrate for casein kinase I (CKI)-α-mediated hyperphosphorylation 85,87 . A regulatory role for NS5A phosphorylation has long been suspected, and was supported by observations that adaptive mutations 7 , engineered mutations 88 , or kinase inhibitors 89 that decrease hyperphosphorylation increase RNA replication but abolish HCV infectivity in chimpanzees 90 .…”

“…The flavivirus genome does not encode a homologue of NS5A; its NS5 protein has RNAdependent RNA polymerase (RdRp) and methyltransferase activities, which are important for RNA synthesis and genome capping respectively ( Fig 5). Despite no sequence similarity to NS5A, NS5 has a zinc-binding motif 96 and is phosphorylated by a putative NS5A-kinase 86 , suggesting functional homology between these proteins 86 . Evidence that DEN NS5 localizes and interacts with NS3 only when hypophosphorylated implies that this protein might also mediate a phosphorylation-dependent relocalization, or disruption, of the replicase complex 97 .…”