The lysosomal LAMTOR-Rag axis functions as a checkpoint for antiviral interferon production

Lysosomes, best known for their nutrient-sensing and degradative roles, have emerged as essential hubs for antiviral defense. While higher organisms depend on type I interferon (IFN) induction for antiviral responses, the mechanism by which lysosomal signaling enables IFN-β production remains unclear. Here, we identify an evolutionarily repurposed lysosomal pathway—centered on the LAMTOR-Rag GTPase complex—that governs IFN-β production through dual transcriptional and post-transcriptional regulation. Genetic ablation of LAMTOR or Rag GTPases in macrophages abolishes IFN-β responses despite intact pattern recognition receptor (PRR) signaling, uncovering a lysosome-specific checkpoint essential for antiviral immunity. Mechanistically, Rag GTPase activity controls IRF expression to prime IFN transcription, while upon PRR stimulation, the tumor suppressor FLCN recruits p38 MAPK to lysosomes, where Rag-dependent p38 phosphorylation stabilizes Ifnb1 mRNA. Nutrient availability dynamically modulates Rag nucleotide states, linking IFN production to metabolic capacity. Notably, this checkpoint operates independently of mTORC1, illustrating how an ancient nutrient-sensing module was co-opted for immune regulation. Disruption of the LAMTOR-Rag–FLCN–p38 axis impairs IFN induction in vitro and antiviral responses in vivo, underscoring its physiological significance. Our findings redefine the lysosome as a central signaling hub integrating metabolic and immune cues, providing evolutionary and mechanistic insights into antiviral defense and potential therapeutic strategies for viral infections and inflammatory diseases.