HSV病毒蛋白调节细胞程序性坏死的分子机制

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.

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。

细胞程序性坏死（Necroptosis）是一种受调控的细胞坏死方式，主要依赖于受体相互作用激酶3（RIPK3）及其底物蛋白混合谱系激酶结构域蛋白（MLKL）的活化。细胞程序性坏死在机体防御病原体中发挥重要作用。人单纯疱疹病毒（HSV）是人类常见的病原体，主要引发口腔和唇等部位感染如疱疹性龈口炎，严重的还会引起脑炎等。我们前期工作发现鼠源RIPK3能识别HSV病毒蛋白R1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RIPK3活化，但其具体机制不详。本项目发现将人源RIPK3的RHIM结构域置换成鼠源RIPK3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。我们发现HSV病毒蛋白 R1(HSV-1 ICP6, HSV-2 ICP10)蛋白诱导人RIPK3发生磷酸化修饰，但不会诱导鼠源RIPK3发生磷酸化修饰，而且位点不同于细胞程序性坏死所需的S227活化位点。HSV R1诱导的RIPK3磷酸化不依赖于RIPK1蛋白的激酶活性，但依赖于RIPK3自身的RHIM结构域。我们发现HSV R1介导的人源RIPK3修饰主要发生于RIPK3第467位的苏氨酸与第513位的酪氨酸。我们进一步研究发现HSV R1介导的人源RIPK3磷酸化修饰位点（第467位苏氨酸与第513位的酪氨酸）对于细胞程序性坏死起着负调控作用。RIPK3第467位的磷酸化位点失活使得人源细胞能够发生HSV-1感染诱导的细胞坏死。而且，我们利用鉴定的RIPK3高效抑制剂发现阻断宿主细胞程序性坏死通路有利于HSV感染的宿主细胞存活以及病毒的繁殖。我们的研究在机制上揭示了HSV病毒蛋白 R1介导的负调控天然宿主人源细胞程序性坏死的机制，在功能上进一步明确了HSV 病毒在进化过程中获得逃逸宿主防御的能力，为深入探索HSV 致病机理和疾病的治疗提供新的思路。