PKR engages viral RNA and intron-retained host transcripts during poxvirus infection.

Recognition of double-stranded (ds) RNA is central to antiviral defense, yet how RNA sensors are activated during infection remains unclear. Here, we demonstrate that during vaccinia virus (VacV) infection, PKR binding is enriched on viral RNAs and host-intron-containing transcripts. During infection, RNase L activation impaired pre-mRNA splicing, promoting accumulation of cytoplasmic intron-retaining transcripts. Small-molecule inhibition of splicing activated PKR independently of RNase L, supporting defective pre-mRNA splicing as a source of PKR ligands. PKR also engaged structured viral RNAs, including telomere-derived species with distinct activating properties. PKR signaling was further shaped by the cellular RNA-binding protein PACT, which enforces a dsRNA abundance threshold for activation, and suppressed by the viral antagonist E3. These findings reveal that PKR monitors both virus-derived and processing-defective host RNAs during infection, establishing cooperativity at the intersection of RNA processing, viral replication, and innate defense.