The Pathogen HSV-1-mediated Negative Regulation Of Necroptosis In Human Host Cells

[Background]When pathogens infect the host cells,the necroptosis of the infected cells is one of the most important mechanisms in host defense against pathogens.Necroptosis is a regulated form of necrosis,which is mediated by the activation of receptor interacting protein kinase 3(RIPK3)and its substrate protein mixed lineage kinase domain like protein(MLKL).Human herpes simplex virus 1(HSV-1)is a common pathogenic herpes virus,which mainly causes oral and lip infections such as herpetic gingivitis,and rarely causes fatal sporadic encephalitis.When HSV-1 infects mouse cells,ICP6 interacts with mouse RIPK3 via their RHIM domains,leading to the activation of mouse RIPK3.The activated mouse RIPK3 further activates the downstream substrate MLKL to mediate necroptosis of infected mouse cells,thus inducing an effective host-defense mechanism against HSV-1.Interestingly,infection of HSV-1 the natural human host cells fails to activate necroptosis in human cells.ICP6 has the ability to bind to human RIPK3.However,the interaction between human RIPK3 and ICP6 leads to the inactivation of human RIPK3,which results in human cells escaping from necroptosis and viral proliferation.Therefore,ICP6 can inhibit and promote necroptosis in human and mouse cells,respectively.The mechanism underlying ICP6-mediated negative regulation of on necroptosis in natural host cells remains to be elucidated.Future understanding of the mechanism through which ICP6 negatively regulates necroptosis in human cells might provide a new way to reveal the pathogenesis of HSV-1.[Objective]The aim of this study is to explore the mechanism underlying negative regulation of necroptosis by HSV-1 viral protein ICP6 in natural host human cells and to understand anti-necroptotic strategies evolved by HSV-1.This study is expected to provide new insights into the pathogenesis of HSV-1.[Methods](1)In order to explore how ICP6 differentially regulates human RIPK3 and mouse RIPK3,we co-transfected ICP6 with human RIPK3 and mouse RIPK3 to examine whether ICP6 could trigger distinct modifications of human RIPK3 and mouse RIPK3.(2)Using the protein purification technology and phosphorylation modification detection system,we determined the ICP6 induced modification of human RIPK3.(3)Molecular cloning was used to construct recombinant plasmids expressing truncated RIPK3 or mutant forms of RIPK3.(4)We used point mutation technique to generate mutant forms of RIPK3 including the candidate phosphorylation sites mutated into A to map the phosphorylation sites in human RIPK3 induced by ICP6.(5)Using lentivirus-expression system,we generated cell lines expressing the mutant forms of RIPK3 to evaluate the impacts of identified RIPK3 phosphorylation sites on necroptosis.[Results](1)ICP6 is found to induce modification of human RIPK3.(2)ICP6-mediated modification of human RIPK3 depends on the RHIM domain of RIPK3,but not on the kinase activity and phosphorylation site S227 of RIPK3.(3)ICP6 induces phosphorylation of human RIPK3 because that ICP6 induced modification was reduced in the presence of phosphatase.(4)ICP6 induces phosphorylation of human RIPK3 at its c-terminal region of 431-518 aa.(5)Threonine 467 and tyrosine 513 are required for ICP6 induced phosphorylation of human RIPK3.(6)T467A(mutation of threonine 467 to alanine)enhances TNF-induced necroptosis as well as the sensitivity of HeLa cells to HSV-1 infection.T467D(mutation of threonine 467 to aspartate),which is expected to mimic the phosphorylation state of RIPK3 at 467,inhibits TNF-induced necroptosis.(7)Y513D(mutation of tyrosine 513 to aspartate),which is expected to mimic the phosphorylation state of RIPK3 at 513,inhibits TNF-induced necroptosis.[Conclusion]To summarize,we have demonstrated that overexpression of HSV-1 protein ICP6 can induce phosphorylation of human RIPK3,but not of mouse RIPK3.ICP6 induces phosphorylation of human RIPK3 at T467 and Y513.Also ICP6-induced phosphorylation of human RIPK3 at T467 and Y513 may account for ICP6-mediated negative regulation of necroptosis in human cells.