# Host Protein Kinase C⍺: The novel Mitogen Activated Protein Kinase (MAPK) specific scaffold regulating nuclear export of influenza virus ribonucleoprotein complexes

## TL;DR

The paper reveals how the host protein PKC⍺ acts as a scaffold to help influenza virus replicate by enabling the phosphorylation of viral proteins.

## Contribution

The study identifies PKC⍺ as a novel MAPK-specific scaffold that stabilizes kinase-substrate interactions critical for influenza virus replication.

## Key findings

- PKC⍺ forms a multiprotein complex with MEK1, ERK2, and viral NP to regulate RNP nuclear export.
- ERK2 phosphorylates the viral nucleoprotein NP, which is essential for virus propagation.
- Dominant negative PKC⍺ blocks complex formation and viral replication.

## Abstract

Host protein kinase C (PKC) isoforms are well known modulators of different steps of influenza virus replication cycle. PKC⍺ was reported to activate the Rapidly Accelerated Fibrosarcoma (Raf)/ Mitogen-activated protein kinase kinase (MEK)/ Extracellular signal-regulated kinase (ERK)- mitogen-activated protein kinase (MAPK) pathway to promote nuclear export of influenza virus ribonucleoprotein complexes (RNPs). However, the molecular mechanism by which PKC⍺ activates specific members of the MAPK cascade and thereby facilitate virus replication, has never been investigated. Here we unravel the novel role of PKC⍺ as a MAPK-specific scaffold to bridge stable kinase-substrate interaction between ERK2 with influenza virus nucleoprotein NP, the major constituent of RNP. Using 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 MAPK cascade, but also participates in stable interactions with the upstream kinase MEK1, effector kinase ERK2, and the substrate NP, thereby forming a multiprotein complex that regulate ERK2 activation, substrate recognition and subsequent phosphorylation events. This multiprotein complex localizes in the nucleus early during infection but eventually moves into cytoplasm at later stages of the viral life cycle. Overexpression of a dominant negative variant of PKC⍺ blocks this complex formation, vRNP export and progeny virus production, thereby establishing PKC⍺ as a key regulator of influenza virus replication. In summary, our results advance the molecular level understanding of the cross-talk between PKC⍺ and MAPK pathway supporting influenza A and B virus replication.

Kinases are key regulators of various signalling cascades. Kinase-substrate interactions are generally transient but can be stabilized by scaffolding or anchoring proteins. This study shows how influenza virus exploits host PKCα as a scaffold protein to activate other cellular kinases (MEK1 and ERK2) and thereby recruit them upon viral replication machinery (RNPs). This RNP associated multi-kinase complex facilitates ERK2 mediated phosphorylation of viral NP protein, which regulates RNP’s transport across nuclear membrane and thereby control 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 could be targeted to develop novel host directed anti-influenza therapy.