Phosphorylation by distinct kinases regulates cardiovirus Leader protein function at the nuclear pore complex

Active nucleocytoplasmic transport is inhibited by the cardiovirus encephalomyocarditis virus (EMCV) Leader (L) protein. L is necessary and sufficient for this inhibition, functioning at the nuclear pore complex (NPC) where it binds RanGTPase, the energy source behind active transport, and causes phosphorylation of nucleoporins (nups), the proteins that compose the NPC. L has no kinase activity, but rather, causes the activation of mitogen activating kinases (MAPKs) that target specific nups. This work aims to determine whether other cardioviruses, specifically Saffold virus (SafV) 2 and Theiler's murine encephalitis virus (TMEV, BeAn strain) L proteins fulfill similar roles at the NPC to EMCV L. These three cardioviruses manifest very differently, and, as L is responsible for much of EMCV's anti-host functions, we hypothesize the differences in disease etiology may be caused by the L protein. Furthermore, L varies the most between EMCV, SafV and TMEV, with the SafV and TMEV L containing additional, functionally uncharacterized domains. Herein we use recombinant protein assays to show that all three cardiovirus Ls inhibit nucleocytoplasmic trafficking and induce the phosphorylation of nups. All three L proteins can bind RanGTPase and the viral protein 2A. Additionally, exportins are bound directly by all three Ls, and MAPKs are activated and bound indirectly. L proteins differ, however, in their phosphorylation state. EMCV L is phosphorylated by casein kinase 2 (CK2) and spleen tyrosine kinase. Interestingly, CK2 does not phosphorylate SafV or TMEV Ls. We identify AMP-activated protein kinase (AMPK) as a kinase that targets SafV and TMEV L proteins. AMPK also acts upon the CK2 site in EMCV and is activated by viral infection. Mutation of L's phosphorylation site results in reduced nup phosphorylation and AMPK and MAPK activation. This work suggests a model in which cardiovirus L protein binds 2A, trafficks to the NPC, dissociates from 2A, becomes phosphorylated (increasing its affinity for exportins), and binds a complex of RanGTPase, exportin, and one or more MAPKs. This complex phosphorylates nups, thereby inhibiting nucleocytoplasmic transport. Abundance and activity of L-targeting kinases may therefore be the limiting factor in L activity, manifesting a difference in cardiovirus L function.