Epstein-Barr virus glycoproteins and virus spread

描述(申请人提供)：爱泼斯坦-巴尔病毒(EBV)由全球95%以上的成年人携带，与免疫母细胞淋巴瘤、伯基特淋巴瘤、霍奇金病、鼻咽癌和胃癌的发生有关。疾病的近因是潜伏感染细胞的行为。然而，有效的复制对病毒在主机内和主机之间的传播至关重要，并影响病毒负载。病毒复制增加的血清学证据是一些EBV相关肿瘤发生的危险因素。这项研究的长期目标是了解EBV膜蛋白在趋向性和复制效率中所起的作用。该应用侧重于了解糖蛋白gB和gNgM以及包膜蛋白BFRF1的多种功能，以及它们在病毒入侵或传播中所起的作用。糖蛋白gB参与了病毒的组装，对病毒细胞融合很重要；缺乏gn的病毒不表达GM，并且在组装和穿透过程中存在结合缺陷，可能还有衣壳和包膜的解离。BFRF1与BFLF2相互作用，可能起到退出细胞核的作用。酵母双杂交筛选揭示了gB的细胞质尾巴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将通过确定这些相互作用是否可以生化复制，绘制GB上的结合位点图，以及确定结合位点突变(S)是否复制缺乏GB的病毒的表型来测试这些相互作用的意义。这将通过拯救以反式表达的突变的gB的阴性病毒或通过在新衍生的EBV-Bac中构建突变来实现。目的2将验证这一假设，即gB在病毒粒子与初级包膜和外核膜的融合中起作用。含有阻止病毒细胞融合的细胞质尾部突变的GB，或含有胞外结构域突变的GB，将检查是否有能力将病毒从细胞核输送到细胞质。AIM 3将检查一种缺乏BFRF1或其伴侣BFLF2的病毒的表型。AIM 4将比较一种缺乏GM基因的病毒和一种缺乏GM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GM的长细胞质尾巴相互作用可能有助于gNgM-病毒表型的蛋白质，并将检测缺少GM细胞质尾巴的病毒的表型。我们将研究GM对其他糖蛋白募集的影响。

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.