The Regulation Of Heat Shock Binding HBP21 In The Innate Immune Response To Viral Infection

Innate immunity is the first line of immune defense of the human body.When pattern recognition receptors(PRRs)recognize pathogens,innate immunity is initiated.Interferon is produced through autocrine or paracrine,and downstream signaling pathways are activated to produce ISGs which fight against viral infection.HBP21 is the binding protein of HSP70.HBP21 is high in normal hepatocytes,while its expression is severely reduced in liver cancer tissues.Tumors are closely related to immunity.Recent studies show that tumor suppressor genes play an important role in the innate immune response to viral infection.The relationship between HBP21 and immunity is unknown.The regulation of HBP21 in the innate immunse to viral infection were studied in the study.Firstly,virus infection induces the expression of HBP2.Virus-induced interferon can induce interferon-stimulated genes(ISGs)fighting against viral infection.The induction of HBP21 by viral infection relies on IFN receptors,So HBP21 is one of ISGs.Some ISGs often play an important role in the innate immune response to viral infection.HBP21 promotes the innate immune response triggered by viral nucleic acid analog or viral infection,as demonstrated in the third chapter.The mechanisms has been explored in the followed chapters.The fourth chapter mainly demonstrated the signaling pathways and key signaling proteins that play important roles in the promotion of the innate immune response to viral infection by HBP21.HEK293 T cells were co-transfected with the plasmid encoding HBP21 with the plasmid encoding different transcriptional factor such as NF-k B,IRF7,or IRF3.The data showed that HBP21 enhances the activity of IRF3-activated IFN promoter,indicating that HBP21 promotes the innate immune response through the activation of IRF3 signaling pathway.HBP21 enhanced the activation of phosphorylated IRF3 triggered by RNA or DNA viral infection.The result was confirmed in primary macrophages isolated from HBP21 knockout mice.Consistently,HBP21 increased the formation of IRF3 dimer and its distribution in the nucleus in the cells.Further study showed that HBP21 targeted IRF3 protein in the IRF3-mediated signaling pathway and interacted with the IAD region of IRF3.The fifth chapter mainly showed the mechanism of HBP21 promoting the activation of IRF3.Since HBP21 has TPR structure,proteins containing TRP structure often regulate the interaction between protein and protein.As expected,HBP21 enhanced the interaction between TBK1 and IRF3.The online software predicts that HBP21 might be phosphorylated at Ser153.HBP21 is highly conserved in evolution.The amino acid sequence alignment from different species shows that Ser85 and Ser153 of HBP21 are highly conserved.Our study showed that viral infection triggered the phosphorylation of HBP21 at Ser85 and Ser153.Phosphorylation of HBP21 at Ser85 and Ser153 is necessary for HBP21 to promote IRF3 phosphorylation,dimer formation,and the interaction between TBK1 and IRF3.Based on the structural analysis,it was found that the phosphatase PP5 looks like the structural fusion of HBP21 and the phosphatase PP2 A of IRF3.The TPR structure of PP5 is combined with the conserved phosphatase activity region of its family member,which makes the phosphatase activity of PP5 in an auto-inhibitory state.This study infers that HBP21 may inhibit the phosphatase effect of PP2 A on IRF3.HBP21 indeed inhibits the dephosphorylation of IRF3 by PP2 A,promoting the innate immune response triggered by IRF3.Mechanistically,HBP21 competes with PP2 A to bind to IRF3 by inhibiting the interaction between PP2 A and IRF3.This study elucidates dual mechanisms by which HBP21 promotes the activation of IRF3.Finally,the role of HBP21 in the antiviral innate immune responses was confirmed in HBP21 knockout mice and primary immune cells isolated from these mice..HBP21-knockout mice and wild-type mice were simultaneously infected with the virus acutely.The data showed that HBP21-knockout mice were more susceptible to viral infection and had a higher mortality rate,compared with the wild-type mice.The virus titers in the liver,lung and spleen tissues were higher and the inflammation is more severe in HBP21-knockout mice,compared with the wild-type mice.All the data supported that HBP21 plays an important role in the antiviral activity at physiological levels and HBP21 promotes the innate immune response to viral infection in vivo.In summary,HBP21 enhances the activation of IRF3 by promoting the binding of phosphokinase TBK1 to IRF3 and inhibiting the dephosphorylation of IRF3 by phosphatase PP2 A,thereby promoting the innate immune response to viral infection and antiviral activity.This study provides the novel mechanism of regulating the activation of IRF3,adding a new perspective on the regulation of multiple networks specified on tumor and immunity by IRF3.