The Mechanisms Of Porcine Cold-inducible RNA-binding Protein In Regulation Of Host Innate Immunity

Cyclic GMP-AMP synthase(cGAS)is the key intracellular DNA receptor that recognizes DNA and mediates the synthesis of cyclic GMP-AMP(c GAMP),which induces activation of stimulator of interferon genes(STING)and downstream antiviral immune signals,eventually leading to expression of type I interferon(IFN)and interferon-stimulated genes(ISGs).At the same time,hyperactivation of cGAS may contribute to the occurrence of various autoimmune diseases.Therefore,cGAS activation is tightly controlled by various host factors and mechanisms.While the regulatory mechanisms of human and murine cGAS activation are extensively studies,little is known about the host factors and the mechanisms that regulates porcine cGAS(pcGAS)activation.In order to identify key host factors that regulate activation of pcGAS,we first screened host proteins that may interact with recombinant pcGAS by using Biacore together with mass spectrometry(MS)analysis.The results showed that cold-inducible RNA-binding protein(CIRBP)was one of previously unknown proteins that likely interact with pcGAS.The interaction between porcine CIRBP(p CIRBP)and pcGAS was then verified by immunoprecipitation and immunofluorescence.To further clarify the role of p CIRBP in regulating cGAS-STING signaling pathway,we expressed p CIRBP in 3D4/21 or PK-15 cells respectively and found that p CIRBP expression could significantly promote DNA-induced expression of type I IFN and downstream ISGs.In consistent with such observation,knockdown or knockout of p CIRBP expression strikingly inhibited DNA-mediated expression of type I IFN and downstream ISGs as well as the activation of TBK1 and IRF3.These results suggests that p CIRBP is a novel positive regulator of host antiviral innate immunity.To know if p CIRBP targets cGAS to activate host antiviral immune responses,we firstly stimulated porcine macrophages with c GAMP,the downstream molecules that synthesized by cGAS.The results showed that knockdown or deficiency of p CIRBP did not affect c GAMP-induced STING-mediated phosphorylation of TBK1 and IRF3 and expression of type I IFN and ISGs,demonstrating that p CIRBP indeed acts on pcGAS to regulate host antiviral immune responses.Mechanistically,p CIRBP interacted with the C-terminal domain(CTD)of pcGAS through its N-terminal domain(NTD).Given that cGAS CTD is the key structural region for DNA binding,we therefore examined whether p CIRBP affected pcGAS binding to DNA.Immunofluorescence and DNA binding assays showed that p CIRBP deficiency weakened the DNA binding ability of pcGAS,suggesting that p CIRBP promotes the cGAS binding to DNA through interacting with cGAS and thus facilitates activation of STING and downstream signals.Consequently,p CIRBP deficiency significantly impaired type I IFN production induced by HSV-1 infection and thus promoted viral replication.In summary,we have identified that p CIRBP is a new host interacting protein of pcGAS.p CIRBP enhances cGAS binding to DNA through interaction with its CTD,promoting activation of cGAS-mediated host antiviral immune responses.These results provide novel regulatory mechanisms of pcGAS and suggest that CIRBP might be a potential target for antiviral drug development.