Influenza virus recruits PKCa-MEK1-ERK2 complex to regulate nuclear export of viral ribonucleoproteins and promote virus replication.

Abstract

Host kinases had long been implicated in regulating various steps of influenza virus replication cycle. Previous studies showed that the protein kinase C (PKC) activated mitogen activated protein kinase (MEK)/ extracellular signal-regulated kinase (ERK) cascade promotes nuclear export of viral ribonucleoprotein complexes (RNPs), by triggering phosphorylation of viral nucleoprotein (NP), the major constituent of RNPs. But, the molecular mechanism by which PKC coordinates with specific members of the MAPK pathway to regulate NP phosphorylation remained obscure. Additionally, the molecular interactions of these kinases with NP and the spatiotemporal dynamics of such interactions have never been investigated. Here we unravel the existence of a tripartite PKC-MEK1-ERK2 complex that associates with influenza virus NP and mediates its phosphorylation during the course of infection. Using an analogue sensitive kinase, we show that ERK2 can directly phosphorylate NP at specific serine-threonine residues, which promote vRNP nuclear export and are indispensable for virus propagation. PKC not only activates the MAP kinase, but acts as a scaffold for mediating stable interactions between MEK1, ERK2 and NP, thereby forming an NP associated multi-kinase complex that facilitate its phosphorylation. This multiprotein complex localizes in the nucleus early during infection but into cytoplasm at later stages and overexpression of a dominant negative PKC blocks this complex formation, NP phosphorylation, vRNP export and progeny virus production. These data not only unravel a complex network of virus-host interactions supporting different influenza A and B virus replication, but also shade light upon an unexplored avenue of PKC mediated regulation MAPK pathway. SignificanceThe substrate specificity of kinases is the key for precise regulation of various signaling cascades and can be governed by direct kinase-substrate interaction or indirect interaction mediated through scaffolding or anchoring proteins. This study shows how influenza virus exploit a well-known cellular kinase, PKC, as a scaffold protein to activate and recruit other cellular kinases, MEK1 and ERK2, upon viral replication machinery, RNPs. This RNP associated multi-kinase complex facilitates ERK2 mediated phosphorylation of viral NP protein, which regulates RNP nuclear export and ultimately the production of new virion particles. This work elucidates an unconventional mechanism of kinase-substrate interaction which is critical for influenza virus replication and hence paves the way towards the development of novel host directed anti-influenza therapy.