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

Lysosomes are emerging as important signaling hubs for antiviral defense, yet how they enable type I interferon (IFN-β) production is 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 and thereby its activation, linking IFN production to metabolic capacity. Notably, this checkpoint operates independently of mTORC1, illustrating how an ancient nutrient-sensing module has been 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 support the role of the lysosome as a central signaling hub integrating metabolic and immune cues, suggesting future directions for potential therapeutic strategies against viral infections.