前期工作中我们发现了一种RIG-I被DNA病毒激活的新机制，即病毒假酶(vGAT)能招募宿主细胞的磷酸核糖基甲酰甘氨酰胺合成酶(FPAS)，诱导RIG-I特异性位点的脱酰胺化。这个结果首次证明了模式识别受体可以被一种酶而不是传统的病原体的结构组分或复制中间体所激活，开辟了一个通过脱酰胺化修饰调节蛋白质功能的新领域。这项工作带来了许多科学问题：这种修饰对蛋白质功能的调节是否具有普遍性，脱酰胺化酶的作用机制是什么，脱酰胺化酶是怎样被调节的，等等。运用分子生物学和蛋白质组学方法，通过在其他的疱疹病毒中分析RIG-I的脱酰胺化，研究其他病毒因子和宿主酶以及相关的作用机制，以及鉴定PFAS-vGAT脱酰胺酶的作用底物等研究工作，我们希望能够回答这些问题。而与国内专家的合作，我们期待在脱酰胺化修饰蛋白质功能这个领域有更多突破，为干预病毒感染和疾病治疗带来新的思路。

To persist within an immune competent host, a virus is under constant pressure to evade host immune defense. Herpesvirus is prone to establish life-long persistent infection and deploys an arsenal of weapons to manipulate host immune response. In studying the murine gamma herpesvirus 68 (MHV68), we discovered a novel mechanism of RIG-I activation by DNA viruses. Specifically, viral pseudo enzymes of gamma herpesviruses, known as viral homologues of glutamine amidotransferase and thus named vGAT, recruit cellular phosphoribosylformylglycinamidinesynthetase (PFAS) to induce site-specific deamidation of RIG-I. Three specific deamaditions within RIG-I enabled RIG-I activation independent of viral RNA. These results, for the first time, demonstrate that a pattern recognition receptor is activated by an enzyme, in contrast to the conventional activation mechanism by structural components or replication intermediates of pathogens. Our study also identifies the first protein deamidase in metazoan, opening a new field addressing the regulatory roles of deamidation that governs protein function post-translationally..Our pioneering work brought more questions than answers pertaining to protein deamidation. The first question is whether protein deamidation is a ubiquitous mechanism in regulating fundamental biological processes. We will address the question by probing the RIG-I deamidation in other herpesviruses, particularly herpes simplex viruses. We will identify viral factors and cellular enzymes that collectively deamidate RIG-I and characterize their molecular action during herpesvirus infection. Moreover, we will employ an unbiased, proteome-wide screen to identify other protein substrates of the PFAS-vGATdeamidating enzyme in herpesvirus infection. To achieve this, we will devise both protein affinity purification and biotin ligase-mediated purification, coupled with biochemical analysis, to identify cellular and viral substrates of protein deamidation and characterize the consequence of deamidation on these substrates. The second, but closely related question is how deamidase is regulated. Here we have an excellent example wherein cellular PFAS is enabled to deamidate RIG-I, in the presence of vGAT or viral infection. We will investigate the possible mechanism that herpesviral vGATs activate PFAS, potentially establishing the first regulatory mechanism governing protein deamidation. Finally, to facilitate our detection of deamidated proteins, we will attempt to generate deamidation-specific antibodies that have not been reported. Preliminary work provides feasibility for the development of antibodies reacting with deamidated proteins. Overall, via collaboration with Prof. Zanxian Xia at Central South University，the proposed work will advance our understanding in protein deamidation technically and conceptually.

前期工作中我们发现了一种RIG-I被DNA病毒激活的新机制，即病毒假酶(vGAT)能招募宿主细胞的磷酸核糖基甲酰甘氨酰胺合成酶(FPAS)，诱导RIG-I特异性位点的脱酰胺化。这个结果首次证明了模式识别受体被激活的一种新机制，开辟了一个通过脱酰胺化修饰调节蛋白质功能的新领域。该项目拟在前期工作的基础上，对其他疱疹病毒诱导是否诱导的RIG-I的脱酰胺化，PFAS在病毒感染和天然免疫应答中的新底物，及禽流感病毒蛋白的脱酰胺化修饰等方面开展深入研究。目前工作已取得重要进展，发现人单纯疱疹病毒-1型（HSV-1）的被膜蛋白UL37蛋白是一种脱酰胺酶，能够对蛋白质进行脱酰胺化修饰。UL37与胞质中的模式识别受体-维甲酸诱导基因 I（RIG-I）发生相互作用，导致RIG-I在两个位点发生脱酰胺化，从而阻断RIG-I受体通路介导的抗病毒天然免疫反应。论文“A Viral Deamidase Targets the Helicase Domain of RIG-I to Block RNA-Induced Activation” 于2016年12月在Cell Host＆Microbe，相关工作被《中国病毒学（英文版）》编辑部撰文进行了介绍。这是在病毒中发现的第一个脱酰胺化酶，也阐明了疱疹病毒蛋白通过脱酰胺化修饰拮抗宿主天然免疫的一种新机制，对于相关研究领域的工作具有十分重要的理论指导意义。同时我们也对vGAT诱导PFAS脱酰胺化而激活PFAS的机制，PFAS在病毒感染和天然免疫应答中的新底物的筛选，以及禽流感毒株H9N2病毒蛋白的脱酰胺化修饰的鉴定等方面开展研究，取得了较好进展，并且发现重要的模式识别受体分子cGAS也是UL37的脱酰胺化底物。相关后续工作正在深入进行中。通过与国内专家的合作，我们期待在脱酰胺化修饰蛋白质功能这个领域有更多突破，为干预病毒感染和疾病治疗带来新的思路。