RLR Signaling Research Articles

Proteomics decode RLR and TLR signaling circuitry (136.5)

Abstract RIG-I-like receptors (RLRs) and Toll-Like Receptors (TLRs) play a critical role in sensing microorganisms and activating the early innate immune response. The signaling circuit of innate immunity remains incompletely understood. To systematically investigate the network, we initiated a global proteomic analysis of protein complexes found in the innate immunity pathway. Fifty-five genes involved in TLR and RLR pathways were tagged with FLAG epitopes and stably transfected into 293 cells engineered to express TLR3 or TLR4. After treatment with or without poly(I:C), LPS, or poly(dA:dT), cells were collected to purify the tagged protein complexes. Proteins in each complex were identified by mass spectrometry and data were statistically analyzed. Protein interaction networks were established using Cytoscape software. Sixty-five known interactions were confirmed and seventy-nine new interactions were detected.

LGP2 is a positive regulator of RIG-I– and MDA5-mediated antiviral responses

RNA virus infection is recognized by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), RIG-I, and melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm. RLRs are comprised of N-terminal caspase-recruitment domains (CARDs) and a DExD/H-box helicase domain. The third member of the RLR family, LGP2, lacks any CARDs and was originally identified as a negative regulator of RLR signaling. In the present study, we generated mice lacking LGP2 and found that LGP2 was required for RIG-I- and MDA5-mediated antiviral responses. In particular, LGP2 was essential for type I IFN production in response to picornaviridae infection. Overexpression of the CARDs from RIG-I and MDA5 in Lgp2(-/-) fibroblasts activated the IFN-beta promoter, suggesting that LGP2 acts upstream of RIG-I and MDA5. We further examined the role of the LGP2 helicase domain by generating mice harboring a point mutation of Lys-30 to Ala (Lgp2 (K30A/K30A)) that abrogated the LGP2 ATPase activity. Lgp2 (K30A/K30A) dendritic cells showed impaired IFN-beta productions in response to various RNA viruses to extents similar to those of Lgp2(-/-) cells. Lgp2(-/-) and Lgp2 (K30A/K30A) mice were highly susceptible to encephalomyocarditis virus infection. Nevertheless, LGP2 and its ATPase activity were dispensable for the responses to synthetic RNA ligands for MDA5 and RIG-I. Taken together, the present data suggest that LGP2 facilitates viral RNA recognition by RIG-I and MDA5 through its ATPase domain.