| Virus infection greatly threatens human health.The host innate immune system is the first line to defense against viral infections.In response to virus infection,pattern recognition recptors(PRRs),such as RIG-I-like receptors(RLRs),Toll-like receptors(TLRs),c GAS,etc.,in the host,recognize the pathogen-associated molecular patterns(PAMPs)of the infected viruses,activate innate immune response signaling pathways and induce the transcription of the antiviral cytokines,particularly interferons(IFNs),to resist viral invasion.In addition,as one of the key PPRs of the host,protein kinase R(PKR)also can recognize the viral ds RNA upon viral infections,which is essential for host response to viral infections.Activated PKR phosphorylates the downstream translation initiation factor e IF2α,by which its activity for initiating host global protein translation is inhibited,leading to the host antiviral protein synthesis repression and contributing to viral escape of the immune surveillance.Post-translational modifications(PTMs)regulate multiple physiological and pathological processes in the host,such as cell cycle,DNA damage repair,cell metabolism,immune homeostasis,and cell signaling.Among them,ubiquitination is one of the important PTMs.The regulation of the transcription of the cytokines by ubiquitination has been widely studied in recent years,however,the role of ubiquitination modification in regulating the PKR-e IF2α signaling pathway remains unclear.TRIM21 is a member of TRIM superfamily and has been reported to associate with autoimmune diseases(such as systemic lupus erythematosus)and tumor development.Our previous study showed that TRIM21 promotes innate immune response by positively regulating the RLR signaling pathway.However,whether TRIM21 is able to regulate the PKR signaling pathway is unknown.Based on this,this thesis focuses on to investigate the mechanism of TRIM21 in regulating stress-induced activation of the PKR signaling pathway:Firstly,the role of TRIM21 in the activation of PKR signaling pathway was explored.Knockout or knockdown of TRIM21 promoted the PKR activation induced by the classical PKR agonist poly(I:C)or viral infections.And,as a consequence,the intracellular protein synthesis was dramatically inhibited by TRIM21 deficiency,indicating that TRIM21 inhibits the activation of PKR signaling pathway.(Chapter 2)Secondly,the mechanism of TRIM21 inhibiting the activation of PKR signaling pathway was investigated.Co-IP experiments revealed that TRIM21 interacts with PKR but not e IF2α,indicating that TRIM21 targets PKR.However,TRIM21 did not directly ubiquitinate PKR,but the E3 ligase activity of TRIM21 was crucial for PKR inactivation,suggesting that TRIM21 may inhibit PKR activation via an indirect manner.Further studies found that TRIM21 has no effect on the ds RNA binding to PKR and PKR dimerization,speculating that TRIM21 may affect the phosphorylation process of PKR;We found that TRIM21 interacts with PKR phosphatase PP1α,and viral infection facilitated their interaction,indicating that TRIM21 inhibits PKR activation through PP1α.Further study revealed that TRIM21 can catalyze K6-linked ubiquitination of PP1α at Lysine60(K60),and viral infection enhanced PP1α ubiquitination mediated by TRIM21.TRIM21 lost the function to inhibit PKR activation by silencing PP1αor mutating the ubiquitinated site of PP1α.At the same time,the interaction between PKR and PP1α was weakened upon virus infection by mutating the PP1αubiquitination site,which demonstrated that TRIM21 mediated-ubiquitination of PP1α promotes the interaction between PP1α and PKR,resulting in PKR activation inhibition.(Chapter 3,4,5and 6)Finally,we found that TRIM21 resists viral invasion by reversing the antiviral genes protein synthesis inhibition mediated by PKR.Knockdown of the key molecules,such as RIG-I,IRF3,and IFNAR1,in the innate immune signaling pathway,TRIM21 still can resist viral invasion,indicating that TRIM21 can inhibit viral infection via an IFN-independent manner.However,the antiviral role was lost as well as knockdown the expression of PKR,indicating that TRIM21 resists virus invasion by inhibiting the activation of PKR signaling pathway.Furthermore,several antiviral genes regulated by PKR-TRIM21 axis were obtained by using proteomic analysis,some of which the antiviral function was further confirmed.(Chapter 7 and 8)In summary,viral infection promotes the interaction between TRIM21 and the PKR phosphatase PP1α,which results in K6-linked ubiquitination of PP1α by TRIM21.The ubiquitinated PP1α enhances its interaction with PKR and inhibits PKR activation,leading to the reversion of PKR-dependent protein synthesis of host antiviral factors inhibition,restricting viral infection.Collectively,in this study,we investigate the mechanism of TRIM21 negatively regulates PKR signaling pathway and reveal the mechanism of TRIM21 resistant to viral invasion by regulating the protein synthesis,which not only deepens our understanding of the antiviral role of TRIM21,but also provides a new insight for host resistance to viral invasion.Investigation of the mechanism of regulation of host factor protein synthesis in host antiviral activity will broaden our understanding of host resistance to virus invasion,provide a theoretical basis for understanding the key scientific issue of virus-host interaction,as well as provide new targets and strategies for prevention and control of virus infection and vaccine development. |
