Study On The Effect And Regulatory Mechanism Of Programmed Necrosis In Host Defense Against Pathogens

Background and significance: Pathogens induced cell death is a significant portion of mammalian host-defense system. It has been found that RIP3-dependent programmed necrosis help host resist the invasion of pathogens, but the detail mechanisms that how the pathogens activate the RIP3-dependent necrosis remain unclear.Objective: To explore the mechanism of programmed necrosis induced by HSV-1 and how it involved in host defense against virus.Methods: To screen virus which can induce RIP3-dependent necrosis, we check the response of WT and RIP3 knockout(KO) MEFs post-infection with the viruses;To test whether RIP3 deficiency influenced the entry and replication of HSV-1, we measured the expression levels of various HSV-1 proteins and genes between WT and RIP3 KO MEF cells infected with virus by western blot and qPCR;To investigate the cellular requirements for HSV-1 induced programmed necrosis. We infected TNFR1 KO, or TNFR1/TNFR2 double KO, or TLR3 KO MEFs with HSV-1, then checked cell survival rate. To check the role of Nectin-1, CYLD in this pathway, we knocked down Nectin-1, CYLD by RNAi. In order to check whether DAI was involved in the pathway,We generate DAI KO MEF cells by CRISPR/Cas9 approach.To test the role of MLKL in the pathway,we generate MEF cells stably expressing MLKL sh RNA;To explore how HSV-1 activates RIP3-dependent necrosis, we infected RIP3 KO MEFs stably expressing vector or WT RIP3 with HSV-1 respectively, to find the protein interacted with RIP3 by Flag-RIP3 immunoprecipitation and protein mass spectrometry analysis;To test whether ICP6 is able to directly activate RIP3/MLKL-dependent necrosis,we overexpressed ICP6 by retrovirus system;To investigate the role of RIP3 in the control of virus replication in vivo, we infected WT and RIP3 KO mice with the same dose of HSV-1 via i.p. injection. To determined viral titers in serum, brain, spleen, and liver by plaque assay. To determine virus replication in liver, we detected the level of gB by immunohistochemistry.To monitor the mice lethality post infected with HSV-1.Results:(1) HSV-1 infection activates RIP3-dependent necrosis.(2)HSV-1 induced cell death is not apoptosis.(3)RIP3 is unessential for HSV-1 entry, replication.(4)RIP3 is dispensable for virus-induced NF-κB activation.(5)HSV-1 infection-induced necrosis requires Nectin-1 but is independent of TNFR, CYLD and TLR3.(6)HSV-1 infection-induced necrosis is independent of DAI.(7)HSV-1 infection-induced necrosis is dependent on MLKL.(8)ICP6 is involved in RIP3 immunocomplex after HSV-1 infection.(9)ICP6 interact with RIP3 through their RHIM domain.(10)ICP6 is essential for HSV1-1 infection-induced necrosis.(11)ICP6 is sufficient to activate RIP3/MLKL-mediated necrosis.(12)The RHIM domain of RIP3 and ICP6 is required for ICP6 induced necrosis.(13) The RIP3 dependent necrosis helps clearance of HSV-1 replication.(14) RIP3 is critical for host defense against HSV-1 replication and pathogenesis.Conclusion: We first found the molecular mechanisms that HSV-1 protein ICP6 activates RIP3-dependent necrosis which act as a host defensive mechanism.