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

EBV的结构基础附件：CD21

• 批准号: 6917861
• 负责人: JOYCE DIANE FINGEROTH
• 金额: $ 16.33万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-06 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6917861
• 关键词: Epstein Barr virus; X ray crystallography; clinical research; complement receptor; cyclization; human tissue; laboratory mouse; laboratory rabbit; monoclonal antibody; peptide chemical synthesis; protein binding; protein protein interaction; protein purification; protein structure function; structural biology; surface plasmon resonance; tissue /cell culture; virus antigen; virus protein; yeasts

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.

描述(申请人提供)：爱泼斯坦-巴尔病毒(EBV)是一种主要的人类病原体，与多种疾病(淋巴和上皮性肿瘤、急性传染性单核细胞增多症、口腔毛状白斑以及可能的某些自身免疫性疾病)有关。全球90%以上的成年人受到感染。预防病毒传播将对国际健康产生重大影响；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。EBV与其细胞表面附着蛋白CD21相互作用的高度特异性表明，基于CD21的重组蛋白疫苗可能被证明是非常有效的。然而，尽管EBV受体(CR2/CD21)早在20年前就被发现，但CD21与其EBV配体gp350/220相互作用的结构基础仍不清楚。 这项拟议的研究的目的是在原子分辨率上了解这种人类肿瘤病毒与其细胞受体CD21之间的相互作用。这些数据显然对于设计能够中断病毒结合的最佳试剂是必要的。我们的分析将揭示对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封闭剂和免疫原提供一个平台，以刺激对EBV的高度保护性免疫反应。在前期工作中，我们已经确定了CD21的EBV附着区的高分辨晶体结构，并通过功能实验和分子模拟确定了CD21上的关键EBV结合表位。然而，目前还没有关于主要的EBV GlvcoProtein gp350/220的结构数据，gp350/220是一种不寻常的膜抗原，与之前描述的任何蛋白质都缺乏相似性。我们很清楚，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gp350/220：CD21复合体。 在具体目标1中，我们将对EBV附着蛋白gp350的功能部分进行结晶和结构分析。在具体目标2中，我们将生产与CD21和EBV 350/220的复合物用于结构分析(如果没有1，这可能会成功)。在特定的目标3中(独立于1和2的目标)，我们将通过我们的模型来确定模拟gp350相互作用中所涉及的CD21表位的环肽是否能够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。相关的受体和配体蛋白将在毕赤酵母和哺乳动物细胞中表达。将利用分子生物学、蛋白质表达和生物化学、X射线结晶学以及哺乳动物细胞培养和分析(病毒结合和感染分析)技术。这里提出的研究将确定EBV产生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可以防止EBV感染和传播的药物的设计提供坚实的基础。