The Structural basis of EBV Attachment: CD21 & gp350/220

描述（由申请人提供）：EB病毒（EBV）是一种主要的人类病原体，与多种疾病（淋巴和上皮肿瘤、急性传染性单核细胞增多症、口腔毛状白斑病和可能的某些自身免疫性疾病）有因果关系。全世界有90%以上的成年人受到感染。预防病毒传播将对国际卫生产生重大影响;然而，复杂的生命周期和病毒癌蛋白的存在使开发减毒疫苗的努力变得非常复杂。EB病毒与其细胞表面附着蛋白CD 21之间相互作用的高度特异性表明，基于CD 21的重组蛋白疫苗可以证明是非常有效的。然而，尽管EBV受体（CR2/CD 21）在近20年前被鉴定，但CD 21与其EBV配体gp 350/220相互作用的结构基础仍然未知。 这项研究的目的是在原子分辨率下了解这种人类肿瘤病毒与其细胞受体CD 21之间的相互作用。这些数据对于设计能够中断病毒结合的最佳药剂显然是必要的。我们的分析将揭示EBV附着最关键的表位，并定义伴随结合的结构变化。这些信息将为合理设计阻断剂和免疫原提供一个平台，这些阻断剂和免疫原刺激对EBV的高度保护性免疫应答。在初步工作中，我们已经确定了CD 21的EBV附着区的高分辨率晶体结构，并通过功能实验和分子建模，我们已经确定了CD 21上的关键EBV结合表位。然而，没有结构数据在所有的主要EBV糖蛋白gp 350/220，一个不寻常的膜抗原，缺乏任何以前描述的蛋白质的相似性。我们很清楚，如果没有这些信息，这是一个高风险的项目。因此，我们寻求探索性资金（R21）来开发必要的数据，并继续结晶gp 350/220：CD 21复合物。 在具体目标1中，我们将对EBV附着蛋白gp 350的功能部分进行结晶和结构分析。在Specific Aim 2中，我们将与CD 21和EBV 350/220产生复合物用于结构分析（这可能在不存在1的情况下成功）。在特定目标3中，（独立于1和2的目标）我们将确定通过我们的模型模拟涉及gp 350相互作用的CD 21表位的环肽是否可以在体外阻止病毒附着。如果成功，未来的研究将在体内测试这些配体。相关受体和配体蛋白将从毕赤酵母（酵母）和哺乳动物细胞表达。将利用分子生物学、蛋白质表达和生物化学、X射线晶体学以及哺乳动物细胞培养和分析（病毒结合和感染测定）技术。本文提出的研究将定义EBV生产性细胞附着的结构要求，并将评估干预这种相互作用的策略，从而为期待已久的免疫原和预防EBV感染和传播的药物的设计提供坚实的基础。

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is a major human pathogen that has been causally implicated in a broad spectrum of disease (lymphoid and epithelial tumors, acute infectious mononucleosis, oral hairy leukoplakia and possibly certain autoimmune diseases). More than 90% of adults are infected worldwide. Prevention of virus transmission would have a major impact on international health; however, the complex life cycle and the presence of viral oncoproteins have greatly complicated efforts to develop an attenuated vaccine. The highly specific nature of the interaction between EBV and its cell surface attachment protein, CD21, suggests that a recombinant protein vaccine based on CD21 could prove very effective. However, although the EBV receptor (CR2/CD21) was identified almost 20 years ago, the structural basis for the interaction of CD21 with its EBV ligand gp350/220 remains unknown. The objective of the proposed research is to provide an understanding of the interaction between this human tumor virus and its cellular receptor, CD21, at atomic resolution. Such data will clearly be necessary for the design of optimal agents that can interrupt virus binding. Our analysis will reveal the epitopes most critical for EBV attachment and define structural changes that accompany binding. This information will provide a platform for rational design of blocking agents and of immunogens that stimulate a highly protective immune response to EBV. In preliminary work, we have determined a high-resolution crystal structure of the EBV-attachment region of CD21 and through functional experiments and molecular modeling we have identified key EBV-binding epitopes on CD21. However, no structural data at all is available for the major EBV glvcoprotein gp350/220, an unusual membrane antigen that lacks similarity to any previously described protein. We are well aware that without this information, this is a high-risk project. Therefore we seek exploratory funding (R21) to develop the requisite data and to proceed to crystallize the gp350/220:CD21 complex. In Specific Aim 1, we will pursue the crystallization and structure analysis of functional portions of the EBV attachment protein gp350. In Specific Aim 2, we will produce complexes with CD21 and EBV 350/220 for structural analysis (this may succeed in the absence of 1). In Specific Aim 3, (an aim independent of 1 and 2) we will determine whether cyclic peptides that mimic the CD21 epitopes implicated in gp350 interaction by our model can prevent virus attachment in vitro. If successful, future studies will test these ligands in vivo. Relevant receptor and ligand proteins will be expressed from Pichia (yeast) and mammalian cells. The techniques of molecular biology, protein expression and biochemistry, x-ray crystallography as well as mammalian cell culture and analysis (virus binding and infection assays) will be utilized. The research proposed here will define the structural requirements for productive cellular attachment of EBV and will evaluate strategies to intervene with this interaction, thereby providing a solid foundation for the long awaited design of immunogens and of drugs that can prevent EBV infection and spread.