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（由申请人提供）：Epstein-Barr病毒（EBV）是一种主要的人类病原体，与疾病有关（淋巴样和上皮肿瘤，急性感染性单核细胞增多症，口腔毛毛状白细胞和可能某些自身免疫性疾病）的因果关系与广泛的疾病有关。超过90％的成年人被全世界感染。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非常复杂，以开发衰减的疫苗。 EBV及其细胞表面附着蛋白之间相互作用的高度特异性性质CD21表明，基于CD21的重组蛋白疫苗可能非常有效。但是，尽管大约20年前确定了EBV受体（CR2/CD21），但CD21与其EBV配体GP350/220相互作用的结构基础仍然未知。 拟议的研究的目的是在原子分辨率下对这种人类肿瘤病毒与其细胞受体CD21之间的相互作用进行理解。 Such data will clearly be necessary for the design of optimal agents that can interrupt virus binding.我们的分析将揭示对EBV附着最重要的表位，并定义伴随结合的结构变化。该信息将为阻止剂和免疫原的合理设计提供一个平台，从而刺激对EBV的高度保护性免疫反应。在初步工作中，我们已经确定了CD21的EBV连接区域的高分辨率晶体结构，并通过功能实验和分子建模确定了CD21上的关键EBV结合表位。但是，对于主要的EBV Glvcoprotein GP350/220，根本没有结构数据，这是一种与任何先前描述的蛋白质相似的不寻常的膜抗原。我们很清楚，如果没有这些信息，这是一个高风险项目。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。 在特定目标1中，我们将追求EBV附着蛋白GP350功能部分的结晶和结构分析。在特定的目标2中，我们将与CD21和EBV 350/220生产复合物进行结构分析（在没有1个的情况下，这可能会成功。在特定的目标3（独立于1和2的目的）中，我们将确定模仿CD21表位与我们模型相互作用涉及的CD21表位的环状肽是否可以防止体外病毒附着。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以肉芽素（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（病毒结合和感染分析）的技术。此处提出的研究将定义EBV生产性细胞附着的结构要求，并评估策略以干预这种相互作用，从而为期待已久的免疫原子设计和可以防止EBV感染和扩散的药物提供了坚实的基础。