PERK inhibition rewires translational and CMGC protein kinase networks into an antiviral state

Protein kinases (PKs) are central regulators of cellular signaling, yet only a small fraction of the human kinome is targeted therapeutically, and kinase–substrate relationships remain incompletely defined. Here, we systematically characterize kinome regulation during human coronavirus 229E (HCoV-229E) infection across transcriptomic, translational, proteomic, and phospho-proteomic layers. We reveal that pharmacological inhibition of the ER stress sensor kinase PERK reprograms host protein biosynthesis and phospho-proteomic landscapes, simultaneously blocking viral nucleocapsid phosphorylation and modulating multiple host kinases. This rewiring antagonizes virus-induced translational shutdown, along with pronounced regulation of the CMGC kinase family, a pattern conserved in SARS-CoV and SARS-CoV-2 infected cells. Comparative analyses with PERK depletion distinguish on-target from off-target effects of PERK inhibition. Our findings uncover the kinome-scale consequences of PERK perturbation in coronavirus infection and demonstrate how the polypharmacology of PERK inhibitors can be harnessed to establish a potent antiviral state, revealing new avenues for host-directed antiviral strategies.