Project summary The innate immune system employs germline-encoded pattern recognition receptors (PRRs) to detect common pathogen-associated molecular patterns. However, only several PRR signaling pathways, including RIG-I- MAVS, cGAS-STING, and TLR3/4-TRIF, activate interferon regulatory factor 3 (IRF3). Recent studies showed that the innate immune adaptors (MAVS, STING, and TRIF) are phosphorylated at their respective C-terminal consensus motif, pLxIS (p, hydrophilic residue; x, any residue; S, phosphorylation site) by TBK1. The phosphorylation of the pLxIS motif in innate immune adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate type I IFN production. However, whether and how the host or pathogen utilizes this motif to attenuate or block IRF3 signaling is unknown. Our overall objective in this application is to determine the role of the cryptochrome (CRY) proteins in IRF3 signaling pathways and host defense to viral infection. Our preliminary proteomic data showed that cryptochrome (CRY) proteins, CRY1 and CRY2, interacted with IRF3. Interestingly, CRY1 and CRY2 have two and one conserved pLxIE motifs, respectively. As glutamic acid (E) is a phosphorylation mimic, this pLxIE motif might be a constitutively phosphorylated pLxIS. Our pilot studies showed that mutation of E to alanine (A) in the pLxIE abolished the binding to IRF3, suggesting that the pLxIE motif is also a docking site for IRF3. Furthermore, deficiency of both CRY proteins also augmented IRF3 signaling and type I IFN production. However, how CRY proteins inhibit innate immune responses is not well elucidated. Based on the existing literature and our preliminary data, we hypothesize that CRY proteins are decoy adaptors for IRF3 via the pLxIE motif and the binding blocks interaction between IRF3 and innate immune adaptors, thereby inhibiting IRF3 phosphorylation and activation. Aim 1 will determine the role of CRY1/2 in the RIG-I-mediated IRF3 signaling pathway. Aim 2 will dissect the molecular mechanisms of how CRY1/2 suppresses IRF3-mediated innate immunity. Many human immune diseases exhibit circadian rhythmicity in their symptoms and pathology, including asthma, rheumatoid arthritis, and other disorders of the immune system. Our study will bridge the gaps in understanding the role of the circadian proteins CRY1/2 in host innate immune response. This novel mechanism proposed in our application will advance our understanding of the crosstalk between innate immunity and circadian.