Establish and Apply In Vitro System for Human Cytomegalovirus Capsid Assembly

人巨细胞病毒衣壳组装体外系统的建立和应用

基本信息

批准号：
8496701
负责人：
D Wade Gibson
金额：
$ 22.84万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8496701
关键词：
Antiviral Agents Area Bacteriophages Baculoviruses Binding Binding Proteins Biochemical Biological Biological Assay Biological Models CSNK2A1 gene Capsid Capsid Proteins Cells Cessation of life Chimera organism Complement Cysteine Cytomegalovirus DNA DNA Packaging Development Effectiveness Electron Microscopy Fluorescence Future Glycogen Synthase Kinase 3 Herpesviridae Herpesvirus 1 Housing Human Human Herpesvirus 4 Human Herpesvirus 8 In Vitro Individual Infection Knowledge Life MAP Kinase Gene Methods Molecular Morbidity - disease rate Mutation Nature Nuclear Nuclear Envelope Outcome Pathway interactions Peptide Hydrolases Pharmaceutical Preparations Phosphoproteins Phosphorylation Site Pilot Projects Preparation Procedures Process Proteins Reaction Recombinants Relative (related person)Research Research Design Scaffolding Protein Sedimentation process Series Simplexvirus Source Structure Surface System Systems Development Temperature Testing Therapeutic Virion Virus Virus Assembly Virus Diseases Work base cell assembly design experience flexibility gammaherpesvirus high throughput screening mortality mutant novel novel strategies pathogen prevent protein function protein protein interaction prototype research study sarcoma scaffold self assembly success

项目摘要

DESCRIPTION (provided by applicant): We present a plan to establish an in vitro system for studying capsid assembly in the beta-herpesviruses (HCMV). Deficiencies in earlier efforts have been recognized and a strategy to overcome them is developed. There is a need for this system now to answer questions out of reach to more biochemical and biological approaches, and that need is expected to increase as more detailed information is required to develop virus- structure-based therapeutics, and as model systems for herpesvirus tegumentation and DNA packaging evolve. Our research design proceeds methodically from a series of in-cell experiments (Aim 1) that will identify the specific HCMV capsid and capsid-associated proteins required to achieve assembly in recombinant baculovirus-infected cells. As part of those studies, we will investigate the possibility that HCMV capsids require stabilization by a surface-binding protein such as the smallest capsid protein (HCMV pUL48/49), or a putative counterpart of the bacteriophage "cap" protein (HCMV pUL77), or perhaps the tightly capsid-associated basic phosphoprotein (HCMV pUL32), which is without a counterpart among other herpesviruses. We will also establish a cell-free in vitro capsid assembly system for HCMV (Sub-aim 1a), as a complementing and alternate approach to in-cell assembly. We will use the methods pioneered for HSV assembly to guide our work. Not only will an in vitro system substantially increase experimental flexibility, it may prove the most direct and expedient way to identify and overcome shortcomings of the in-cell approach. Recent studies demonstrate the ability of chimeric CMV scaffold proteins to drive assembly of HSV and KSHV capsid shells. In Aim 2, the effect of function-disrupting mutations in the essential HCMV UL80 scaffolding proteins (pUL80.5 and pUL80a) will be tested to assess the suitability of this method as a way to probe the molecular interactions involved in capsid formation. This system allows us to overcome barriers imposed by the essential nature of the scaffolding proteins and the inability to study these functions in HCMV infected cells. The outcome of this R21 proposal will be the development of an in-vitro capsid assembly system for a high-priority human pathogen. The future development of this system to incorporate in-vitro DNA packaging, tegumentation and envelopment of capsids will have significant impact on the development of new and novel antiviral strategies against this virus. HCMV is one of the nine herpesviruses that infect people, and prototypic of the three human beta-herpesviruses. It causes significant morbidity and mortality, especially in connection with transplacental infections and in immuno-compromised groups. New approaches are needed to prevent and treat infections by this and all herpesviruses, and the assembly system to be developed through this project will aid that endeavor.

描述（由申请人提供）：我们提出了一项建立体外系统的计划，用于研究 β-疱疹病毒（HCMV）中的衣壳组装。早期工作中的缺陷已经被认识到，并制定了克服这些缺陷的策略。现在需要该系统来回答更多生化和生物学方法无法解决的问题，并且随着需要更详细的信息来开发基于病毒结构的疗法以及作为疱疹病毒覆盖的模型系统，这种需求预计会增加DNA 包装也在不断进化。我们的研究设计有条不紊地从一系列细胞内实验（目标 1）开始，这些实验将鉴定在重组杆状病毒感染的细胞中实现组装所需的特定 HCMV 衣壳和衣壳相关蛋白。作为这些研究的一部分，我们将研究 HCMV 衣壳是否需要通过表面结合蛋白来稳定，例如最小的衣壳蛋白 (HCMV pUL48/49)，或噬菌体“帽”蛋白的推定对应物 (HCMV pUL77) ，或者可能是与衣壳紧密相关的碱性磷蛋白（HCMV pUL32），它在其他疱疹病毒中没有对应物。我们还将建立 HCMV 体外无细胞衣壳组装系统（子目标 1a），作为细胞内组装的补充和替代方法。我们将使用 HSV 组装首创的方法来指导我们的工作。体外系统不仅会大大提高实验灵活性，而且可能被证明是识别和克服细胞内方法缺点的最直接、最便捷的方法。最近的研究证明了嵌合 CMV 支架蛋白能够驱动 HSV 和 KSHV 衣壳壳的组装。在目标 2 中，将测试必需 HCMV UL80 支架蛋白（pUL80.5 和 pUL80a）中功能破坏性突变的影响，以评估该方法作为探测衣壳形成中涉及的分子相互作用的方法的适用性。该系统使我们能够克服支架蛋白的本质所带来的障碍以及无法在 HCMV 感染细胞中研究这些功能的障碍。 R21 提案的成果将是为高优先级人类病原体开发体外衣壳组装系统。该系统的未来发展将结合体外 DNA 包装、衣壳的覆盖和包封，将对针对该病毒的新型抗病毒策略的开发产生重大影响。 HCMV 是感染人类的九种疱疹病毒之一，以及三种人类β-疱疹病毒的原型。它导致显着的发病率和死亡率，特别是与经胎盘感染和免疫功能低下群体有关。需要新的方法来预防和治疗这种疱疹病毒和所有疱疹病毒的感染，而通过该项目开发的组装系统将有助于这一努力。