CSDE1 promotes viral immune evasion through RNA-dependent and phosphorylation-modulated liquid-liquid phase separation.

RIG-I-like receptors (RLRs) are essential for antiviral innate immunity through sensing viral RNA. Although liquid-liquid phase separation (LLPS) has emerged as a key mechanism in fine-tuning immune signaling, its role in the initial sensing of viral RNA remains poorly understood. Here, we identify the RNA-binding protein CSDE1 as a negative regulator of RLR signaling that facilitates viral immune evasion. Macrophages deficient in CSDE1 exhibit enhanced interferon production, and CSDE1-knockout mice show increased resistance to viral infection. Mechanistically, CSDE1 forms RNA-dependent biomolecular condensates that function as an RNA-sensing checkpoint by sequestering immunostimulatory viral RNAs and shielding them from RLR recognition. Upon viral infection, activated TBK1 kinase phosphorylates CSDE1, leading to condensate disassembly and relief of immune suppression. Furthermore, CSDE1 promotes condensation of the SARS-CoV-2 nucleocapsid protein, cooperatively antagonizing type I interferon responses. Notably, we identify a small molecule that disrupts CSDE1 condensation and potently suppresses viral replication. Together, our findings reveal a phosphorylation-modulated, phase separation-based checkpoint in RLR signaling and establish CSDE1 condensates as a promising therapeutic target against RNA viruses.