Cardiovirus-Mediated PKR inhibition results from nucleocytoplasmic trafficking disruption

Abstract

Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), known as PKR, is a key antiviral kinase activated by double-stranded RNA (dsRNA) typically produced during viral replication. Upon activation, PKR phosphorylates eIF2α, leading to the inhibition of translation and viral replication. However, many viruses have evolved mechanisms to counteract PKR activity. In Cardioviruses, the Leader protein (L), a short peptide cleaved from the N-terminus of the viral polyprotein, not only inhibits PKR but also blocks interferon production and disrupts nucleocytoplasmic trafficking (NCT). L disrupts NCT by recruiting host RSK kinases to the nuclear pore complex (NPC), where RSK phosphorylates FG-nucleoporins, thereby impairing NCT. L mutations that affect NCT disruption also impact PKR inhibition, suggesting a mechanistic link between NCT and PKR activity. Recombinant TMEV and EMCV viruses designed to disrupt NCT through different mechanisms exhibited some extent of PKR inhibition, supporting the link between NCT disruption and PKR inhibition. Immunostaining and live-cell imaging revealed that, during mitosis and after L-induced NCT disruption, a fraction of PKR maps to the nucleoli, where PKR remains inactive despite its recruitment by dsRNA-like structures. This suggests that nucleolar sequestration contributes to PKR inhibition. Additionally, L-mediated NCT disruption leads to the release of nuclear RNA-binding proteins (nRBPs) into the cytosol, which may bind or modify viral dsRNA, further preventing PKR activation. Collectively, these results highlight nucleocytoplasmic trafficking as a critical regulatory mechanism governing PKR activation. Thus, beyond the specific action of cardiovirus L protein, our study reveals that interference with host nucleocytoplasmic transport can significantly impact the subcellular localization and functional regulation of immune effectors such as PKR.