Integrated Sensing of Viral RNA and RNase L-Driven Intron Retention Orchestrates Anti-Poxvirus Immunity

The recognition of double-stranded RNA (dsRNA) is a critical component of antiviral innate immune responses, yet how distinct sensors integrate their activities to enforce restriction remains unclear. Here, we uncover a cooperative mechanism linking the OAS3-RNase L and PKR pathways during vaccinia virus (VacV) infection. Transcriptome-wide analyses reveal that RNase L activation disrupts pre-mRNA splicing, inducing widespread intron retention. These retained introns form dsRNA structures that evade nuclear quality control, accumulate in the cytoplasm, and serve as ligands for PKR. Thus, RNase L reshapes the transcriptome to enhance the generation of immunostimulatory substrates, amplifying PKR activation. In parallel, we identify conserved viral RNAs derived from the VacV telomeres, whose terminal loop architectures directly modulate PKR activity, with distinct species either promoting or dampening its activation. Together, our findings establish that PKR signaling emerges from the integrated detection of host-derived introns and conserved viral terminal structures, revealing innate immune cooperativity at the interface of RNA processing, viral replication, and gene regulation.