Nuclear RPSA triggers R-loop accumulation to induce cell death against RNA viral infection

While RNA viruses evade host immune responses by perturbing transcriptional and translational machinery, the host organism counteracts viral invasion through activation of regulated cell death pathways. However, the precise molecular mechanisms underlying host-initiated cell death activation in response to viral infection remain poorly characterized. By genome-wide screening, we identified ribosomal protein RPSA as a broad inducer of RNA virus replication-dependent cell death that confers antiviral protection. Rpsa conditional knockout mice exhibited heightened susceptibility to RNA viruses with attenuated cell death activation in infected tissues. Mechanistically, RNA virus infection triggers K29-linked polyubiquitination of RPSA, which strengthens its binding to the helicase domain of NAT10 and potently inhibits NAT10's helicase activity, thereby blocking R-loop resolution. The resulting R-loop accumulation drives DNA damage-mediated cell death execution. Our findings uncover a novel intranuclear "inside-out" R-loop-driven cell death pathway orchestrated by RPSA, providing fresh insights into virus-host interactions and identifying potential therapeutic targets for RNA viral infections. CUT&Tag of γ-H2AX in wild-type or RPSA-knockout A549 cells infected with IAV for 12 hours