Regulatory Mechanisms Of Signal Transduction In Antiviral Innate Immunity

The induction of type I IFNs by viral infection has been the hot topics of innate immune responses against viral infection. The host cells recognize pathogen-associated molecular patterns (PAMPs) of pathogen by pathogen recognition receptors (PRRs), which trigger intracellular signaling cascades, leading to the production of type I IFNs, proinflammatory cytokines and chemokines.During viral infection of cells, the viral nucleic acids are recognized by Toll-like receptors (TLRs), such as TLR3,7 and 9, retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), such as RIG-I and MDA5, and recent identified DNA receptors, such as DAI and IF 116. Among the PRRs, the RLRs, RIG-1 and MDA5, exist in cytoplasm and recognize viral RNA. Upon binding to viral RNA, RIG-1 and MDA5 recruit the adapt protein VISA(also known as MAVS,IPS-1 and Cardif). On one hand, VISA interacts with TRAF2 and/or TRAF6 to activate IKK kinase complex, leading to activation of transcription factor NF-κB; on the other hand, VISA is associated with TRAF3 and TBK1 to activate IRF3 by phosphorylation. Then, the activated transcription factors IRF3 and NF-κB enter into the nucleus and collaboratively trigger the expression of type I IFN genes. MITA (also known as STING) is another important adaptor protein, which was recently identified in signaling pathway mediated by RLRs. MITA is also located in mitochondria and constitutively associated with VISA. After viral infection, TBK1 is recruited by the association of MITA and VISA, leading to the formation of VISA-MITA-TBK1-IRF3 complex through self-oligomerization. The complex enhances the phosphorylation and activation of IRF3 mediated by TBK1, triggering the expression of type I IFNs.Recognition of invading virus by host cells has long been a hot research field in the antiviral innate immunity. Although many PRRs have been identified during the last decades, there are still some questions remaining to be answered. In order to search for the unknown molecules which paly roles in the signaling transduction of induction of type I IFNs triggered by viral infection, we screened a gene library by using pGL3-ISRE as a reporter gene. We found that LSM14A activated the ISRE and the IFN-p promoters and markedly increased the expression of type I IFNs induced by Sendai virus (SeV) and herpes simplex virus type (HSV). On the contrary, knockdown of endogenous LSM14A remarkably inhibited early expression of type I IFNs induced by SeV infection and also inhibited activation of the IFN-βpromoter triggered by cytoplasmic dsRNA and dsDNA. Vesicular stomatitis virus (VSV) plaque assay showed that LSM14A effectively inhibited cytoplasmic replication of virus. LSM14A could directly bind to viral RNA and DNA, acted as a new potential cytoplasmic receptor for viral nucleic acids and interacted with RIG-I of VISA to mediate the expression of type I IFNs induced by viral infection.To further investigate how the functions of MITA in virus-induced signaling pathways are regulated, we identified ISG56 as a MITA-associated protein through a biochemical purification approach. ISG56 is one of the first identified proteins induced by viral infection and type I IFNs. We found that overexpression of ISG56 inhibited SeV-triggered activation of IRF3, NF-κB, and the IFN-βpromoter, whereas knockdown of endogenous ISG56 had opposite effects. Consistently, overexpression of ISG56 reversed cytoplasmic poly(I:C)-induced inhibition of VSV replication, whereas knockdown of endogenous ISG56 inhibited VSV replication. Competitive co-immunoprecipitation experiments indicated that ISG56 disrupted the interactions of MITA with up-stream VISA and down-stream TBK1 by binding to MITA. These results suggest that ISG56 is a mediator of negative-feedback regulation of virus-triggered induction of type I IFNs and cellular antiviral responses.Our studies identified LSM14A as a new potential receptor for viral nucleic acids, which is involved in signal transduction of virus-induced production of type I IFNs, which disclosed a new pathogen-recognition mechanism in cellular antiviral immune responses. We also found that ISG56, a protein induced by type I IFNs, negatively regulate virus-triggered type I IFNs production, demonstrating a new negative-feedback regulatory mechanism of antiviral innate immune responses.