HCMV感染对神经前体细胞Notch通路的调控

Congenital human cytomegalovirus (HCMV)infection is the top number one cause of birth defects, primarily central nervous system disorders. Previously we have established the HCMV infected neural progenitor cell (NPC) model, and found that HCMV infection induced NPCs abnormal differentiation.Recently we foud that Notch signal pathway molecules, the molecular switch for NPCs' neurogenesis and stem cell characteristics maintenance, were downregulated by HCMV infection.But which viral protein(s)controls the switch and how they regulate the switch is unknown...The aim of this project is to elucidate the mechanism(s) of Notch signal pathway dowmregulation by HCMV infection in NPCs,so that to bridge the gap between HCMV infection and NPCs abnormal differentiation and shed light into understanding how congenital HCMV infection causes brain developmental disorders. To fulfil this aim the tasks include: ..To identify the specific viral proteins that regulate Notch signal pathway.First, treat the infected NPCs with inhibitors of protein synthesis at different times post infection to specify the accurate phase (time) of infection when the moluecules of Notch signal pathway are downregulated. Then, construct the mutant virus variants, wild type and mutant HCMV gene expressing constructs, infect or transfect NPCs, to identify the specific viral proteins that regulate Notch signal pathway and the roles...To specify the regulation relationship among HCMV protein(s)and Notch molecules. First,transfect NPCs with HCMV gene, detect the effect on Notch molecules. Second,for example targeting RBP-J, to investigate the protein-protein interaction and specify the upstream and downstream molecules by co-immunoprecipitation and RNAi. Then,co-overexpress Notch intracellular domain (NICD), HCMV gene in NPCs, to further specify the regulation relationship among HCMV proteins and Notch molecules...To further map the regulation network of HCMV proteins and Notch molecules. Using the identified viral protein or RBP-J or Sox2 as the bait to screen the preys by yeast-two-hybridization from fetal brain library.Confirm the interaction between the proteins in the NPCs by co-immunoprecipitation and RNAi assay.

先天性HCMV感染是导致出生缺陷的最常见病因，主要表现为中枢神经发育畸形。我们用HCMV感染神经前体细胞（NPCs),发现了HCMV下调Notch通路这一维持NPC干细胞特性和调节神经发生的"开关"，而调控此"开关"的病毒蛋白及其调控机制不清楚。本项目拟用蛋白质合成抑制剂等处理HCMV感染的NPCs,确定下调Notch通路的感染时相；通过病毒感染、表达病毒蛋白干预Notch通路，鉴定调控Notch通路的病毒蛋白；在NPCs中过表达病毒蛋白、NICD，以Notch通路分子RBP-J等为靶标，经免疫共沉淀、RNAi等技术，确定病毒蛋白、Notch通路分子间的互作关系；进一步以鉴定的病毒蛋白等为诱饵，从胎儿脑文库中筛选鉴定互作靶标，展示调控网络。旨在揭示HCMV感染调控Notch机制，填补HCMV感染与NPCs增殖分化异常之间的空缺,为解析先天性HCMV感染致胎儿中枢神经发育畸形机制提供新思路。

先天性人巨细胞病毒（human cytomegalovirus，HCMV）感染导致胎儿神经发育畸形，是出生缺陷最常见的病原性病因，而人神经前体/干细胞（NPCs）HCMV感染的主要靶标。由于HCMV感染严格的种属特异性，对HCMV感染完全容许的NPCs是研究HCMV感染导致神经发育异常的理想细胞模型。但NPCs的细胞特性和HCMV对其感染特性尚不清楚。本课题通过建立NPCs细胞库重建了HCMV感染的NPCs模型，并基于该模型研究了HCMV对NPCs关键通路Notch信号途径的影响。通过分离并鉴定人胎脑NPCs，我们成功地建立了含超过270个不同个体来源的神经干细胞库，发展优化了HCMV感染的NPCs模型，并利用该模型研究HCMV感染对NPCs的影响及机制。.Notch信号通路是维持NPCs的多能性及调控NPCs分化的关键“开关”，我们的研究证明HCMV感染通过异常调节Notch通路而导致NPCs增殖分化异常。该异常调节机制之一是病毒蛋白pp71通过蛋白酶体和溶酶体途径促进Notch通路受体Notch1的活性产物NICD1和配体Jag1的蛋白降解，从而下调NICD1和Jag1的蛋白水平，并引起NICD1和Jag1的细胞定位改变。这一研究揭示了HCMV感染导致神经发育异常的一个可能的新机制。