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

• 批准号: 8385942
• 负责人: D Wade Gibson
• 金额: $ 20.25万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 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/8385942
• 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

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. PUBLIC HEALTH RELEVANCE: Cytomegalovirus (CMV) is one of nine herpesviruses that infect people and cause significant illness and death. New and more effective drugs are needed to treat infections by this virus and one source of targets for developing such drugs is the virus capsid assembly pathway. A model system for studying CMV capsid assembly in vitro will make this a feasible and attractive area to exploit. We propose to establish such a system for human CMV, based on experience, encouraging results from pilot studies, and new knowledge gained from working with the herpes simplex virus prototype system and similarly productive systems for Epstein-Barr virus and Kaposi's Sarcoma-associated herpes virus.

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。现在需要这个系统来回答更多生化和生物学方法无法解决的问题，并且随着开发基于病毒结构的疗法需要更详细的信息，以及随着疱疹病毒覆盖和DNA包装模型系统的发展，这种需求预计会增加。 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 衣壳需要表面结合蛋白稳定的可能性，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)，其没有 其他疱疹病毒中的对应物。 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。 公共卫生相关性：巨细胞病毒 (CMV) 是感染人类并导致严重疾病和死亡的九种疱疹病毒之一。需要新的、更有效的药物来治疗这种病毒的感染，开发此类药物的靶点之一是病毒衣壳组装途径。用于研究巨细胞病毒衣壳组装的体外模型系统将使这一领域成为可行且有吸引力的开发领域。我们建议根据经验、试点研究的令人鼓舞的结果以及从使用单纯疱疹病毒原型系统以及 Epstein-Barr 病毒和卡波西肉瘤相关疱疹病毒的类似生产系统中获得的新知识，建立这样一个人类 CMV 系统。