Epstein-Barr virus glycoproteins and virus spread

描述（由申请人提供）：eb病毒（EBV）在全球95%以上的成年人中携带，与免疫母细胞淋巴瘤、伯基特淋巴瘤、霍奇金病、鼻咽癌和胃癌的发展有因果关系。疾病的近端原因是潜伏感染细胞的行为。然而，高效复制对病毒在宿主内部和宿主之间的传播至关重要，并影响病毒载量。血清学证据表明病毒复制增加是一些ebv相关肿瘤发展的危险因素。本研究的长期目标是了解EBV膜蛋白在趋向性和复制效率中的作用。该应用的重点是了解糖蛋白gB和gNgM以及包膜蛋白BFRF1的多种功能及其在病毒进入或传播中的作用。糖蛋白gB参与病毒组装，对病毒细胞融合很重要；缺乏gN的病毒不能表达gM，并且在装配和渗透过程中，在衣壳和包膜的结合上存在缺陷，也可能存在解离缺陷。BFRF1与BFLF2相互作用，可能在退出细胞核中发挥作用。酵母双杂交筛选揭示了gB细胞质尾部与已知或被认为参与病毒组装的蛋白质之间的相互作用。目的1将测试这些相互作用的重要性，通过确定它们是否可以在生化上复制，绘制gB上的结合位点，并确定结合位点的突变是否会复制缺乏gB的病毒的表型。这将通过挽救以反式表达突变gB的gB负病毒或通过将突变构建为新衍生的EBV-Bac来实现。目的2将验证gB在具有初级包膜和外核膜的病毒粒子融合中起作用的假设。将检查细胞质尾部含有阻止病毒细胞融合的突变或细胞外结构域含有突变的gB，以确定其将病毒从细胞核传递到细胞质的能力。目的3将检查缺乏BFRF1或其伴侣BFLF2的病毒的表型。目标4将比较基因上缺乏转基因的病毒与基因上缺乏转基因的病毒的表型。将进行酵母双杂交和生化分析，以寻找与转基因长细胞质尾部相互作用可能导致gNgM阴性病毒表型的蛋白质，并检查缺乏转基因细胞质尾部的病毒的表型。转基因对其他糖蛋白募集的影响将被研究。

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is carried by more than 95% of the adult population worldwide and is causally implicated in the development of immunoblastic lymphoma, Burkitt's lymphoma, Hodgkin's Disease, nasopharyngeal carcinoma and gastric cancer. The proximal cause of disease is the behavior of a latently infected cell. However, productive replication is critical to spread of virus within and between hosts and impacts virus load. Serologic evidence of increased virus replication is a risk factor for development of some EBV-associated tumors. The long-term goal of this research is to understand the roles that the membrane proteins of EBV play in tropism and efficiency of replication. The application focuses on understanding the multiple functions of glycoproteins gB and gNgM and the envelope protein BFRF1 and the roles they play in virus entry or spread. Glycoprotein gB has been implicated in virus assembly and is important to virus cell fusion; virus lacking gN fails to express gM and has a defect in association and possibly also in dissociation of capsids and envelope during assembly and penetration. BFRF1 interacts with BFLF2 and may play a role in exit from the nucleus. A yeast two-hybrid screen has revealed interactions between the cytoplasmic tail of gB and proteins known or thought to be involved in virus assembly. Aim 1 will test the significance of these interactions by determining if they can be reproduced biochemically, mapping the binding sites on gB and determining if mutation of binding site(s) reproduces the phenotype of a virus that lacks gB. This will be done by rescuing gB minus virus with mutated gB expressed in trans or by building mutations into a newly derived EBV-Bac. Aim 2 will test the hypothesis that gB plays a role in fusion of virions with a primary envelope and the outer nuclear membrane. gB containing mutations in the cytoplasmic tail that block virus cell fusion, or with mutations in the extracellular domain will be examined for the ability to deliver virus from the nucleus to the cytoplasm. Aim 3 will examine the phenotype of a virus that lacks BFRF1 or its partner BFLF2. Aim 4 will compare the phenotype of a virus genetically lacking gM with one that lacks gM. Yeast two-hybrid and biochemical analysis will be done to look for proteins whose interactions with the long cytoplasmic tail of gM may contribute to the phenotype of a gNgM minus virus and the phenotype of a virus that lacks the cytoplasmic tail of gM will be examined. Effects of gM on recruitment of other glycoproteins will be examined.