RIP3/MLKL调控的细胞程序性坏死在机体防御病原体中起重要作用。人单纯疱疹病毒（HSV）是人类常见的病原体。我们前期工作发现鼠源RIP3能识别HSV的R1蛋白启动程序性坏死，从而抑制小鼠体内病毒繁殖，而R1却抑制天然宿主人源细胞中RIP3活化，但其具体机制不详。本项目初期工作发现将人源RIP3的RHIM结构域置换成鼠源RIP3的RHIM促使人源细胞对HSV敏感，并能发生R1诱导的程序性坏死，而且R1蛋白诱导人RIP3发生磷酸化修饰，位点不同于已知的S227活化位点，在此基础上，我们将深入研究人源和鼠源RIP3 RHIM结构域的差异序列在R1调节的程序性坏死中的作用机制，鉴定R1诱导人RIP3磷酸化位点，进而阐明R1负调控人RIP3的机制，还将鉴定可能调节RIP3和MLKL的其他HSV病毒蛋白，并揭示其作用机制，从而充分阐明HSV蛋白对程序性坏死的调节机制，且揭示程序性坏死的负调控机制。

Receptor-interacting kinase3（RIP3）and its substrate MLKL (mixed lineage kinase domain like protein) are core regulators of necroptosis. The elimination of pathogen-infected cells by necroptosis acts as an important host defensive mechanism. Human herpes simplex virus (HSV) is a common human pathogen. Previously, we have demonstrated that RIP3 recognizes HSV protein R1 to activate necroptosis in mouse cells. This process is crucial for the control of HSV replication and pathogenesis in mice. In contrast, HSV infection is unable to induce necroptosis in human cells. Moreover, HSV R1 blocks tumor necrosis factor (TNF)-induced necrosis and RIP3 activation in human cells. The precise mechanism by which the R1 exerts its negative regulatory effect on human RIP3 (hRIP3) has not been fully elucidated. In this proposal, we have found that replacement of the RHIM domain of hRIP3 by the RHIM domain of mouse RIP3 (mRIP3) rendered these human cells sensitive to necroptosis triggered by HSV infection and the ectopic expression of R1. These human cells underwent necroptosis in response to HSV infection or the ectopic expression of R1. In addition, we observed that the expression of R1 induced phosphorylation of hRIP3 independent of hRIP3 S227, which is known to be important for necroptosis. Based on above findings, we will further identify the specific sequence in the RHIM domain of hRIP3 which may negatively regulate hRIP3 activation, and map the phosphorylation site of hRIP3 induced by R1. In addition, we will identify and characterize other HSV proteins associated with hRIP3 or hMLKL during HSV infection and demonstrate how they modulate necroptosis in human cells. This study will fully elucidate how HSV proteins manipulate necroptosis in the natural human host and provide new machinery involved in the negative regulation of necroptosis.