IE1作为E3泛素连接酶在HCMV致胎脑发育畸形中的作用及机制

Neural stem/progenitor cells (NPCs) is the base for fetal brain development. In congenital infection, the main target of human cytomegalovirus is NPCs in the fetal brain. HCMV infection leads to abnormal differentiation of NPCs, which results in maldevelopment of the fetal brain. But the involved pathogenesis remains to be further elucidated. Recently, our team identified, for the first time, the E3 ubiquitin ligase activity of the HCMV immediate early protein IE1, which is also the first identified E3 ubiquitin ligase encoded by HCMV, as well as by beta-herpesvirus family. And Hes1, an essential NPCs fate regulator, has been identified to be the ubiquitinating substrate of IE1. HCMV infection downregulates Hes1 protein level in NPCs through promoting its ubiquitination-proteasomal degradation, and thus dysregulates the proliferation and differentiation of NPCs. Therefore, we hypothesize that the E3 activity of IE1 plays an important role in fetal brain maldevelopment induced by HCMV. To confirm our hypothesis, this project is to: 1) characterize IE1 as the E3 ligase by identifying the catalytic center, finding novel substrate, and revealing its binding site with the substrates or E2; 2) construct the mutants of IE1 protein or HCMV virus with defected E3 activity, and reveal the function of IE1 E3 activity during HCMV infection and replication; 3) finally, understand the role of IE1 as an E3 ligase on NPCs cell fate and fetal mouse brain development. This work will expand and deepen our knowledge of HCMV and virus coded E3 ubiquitin ligases, reveal novel mechanism of HCMV inducing fetal brain maldevelopment, and offer novel potential target for clinical intervention.

神经干细胞（NPCs）是胎脑发育的关键。先天性人巨细胞病毒（HCMV）感染在胎脑内的首选靶标是NPCs，引起其异常分化导致胎脑发育畸形，但具体机制有待深入研究。我们近期鉴定了首个HCMV编码的E3泛素连接酶——IE1，并发现其泛素化底物为NPCs命运的关键调节因子Hes1。HCMV感染NPCs通过IE1促进Hes1泛素化，经蛋白酶体降解使其蛋白水平下调，导致NPCs增殖分化异常。因此，我们推测IE1的E3酶活性在HCMV致胎脑发育畸形中发挥重要作用。为证明该假说，本项目将：1）解析IE1的E3酶特性，鉴定其他泛素化底物、及其与E2酶/底物的结合位点；2）构建E3酶活性缺失的突变蛋白或毒株，揭示IE1作为E3酶对病毒复制和NPCs命运的影响；3）通过小鼠模型研究IE1的E3酶活性对胎脑发育的影响及机制。该研究将拓展HCMV病毒学知识，揭示HCMV致胎脑发育畸形的新机制，为临床干预提供新靶点。

人巨细胞病毒（HCMV）先天性感染会导致胎脑发育损伤，造成严重的神经发育障碍，但目前其具体致病机制尚不清晰。基于我们之前的研究，鉴定了首个HCMV的E3酶——IE1，并发现其泛素化底物为调控NPC细胞命运的关键细胞因子Hes1，此项目在此基础上围绕IE1作为E3酶在胎脑发育中的功能展开。我们完成了以下工作：1. 鉴定CMVs感染下调间隙连接蛋白Cx43。在MCMV先天性感染鼠脑组织样品以及HCMV感染的NPC样品中均发现Cx43被下调；2. 鉴定IE1新的泛素化底物Cx43，证实HCMV-IE1通过泛素化途径下调Cx43；3. IE1调控Cx43依赖直接互作，并通过AA236-250区段与Cx43结合；4. 通过胚胎电转建立外源表达IE1于小鼠皮层中的动物模型，并发现IE1在皮层中下调Cx43，且IE1表达导致胎脑发育异常，表现为皮层变薄，脑室增大等现象； 5. 依赖小鼠胚胎电转模型，跟踪神经元迁移过程，通过阻断IE1和Cx43结合或IE1下调后补偿Cx43两种途径证实IE1通过下调Cx43影响非成熟神经元迁移。此研究拓展对HCMV-IE1的认知，揭示HCMV致胎脑发育畸形的新机制，并为临床干预提供新的靶标。