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）：我们提出了一项计划，以建立一个体外系统，用于研究β-疱疹病毒（HCMV）中的衣壳组件。早期努力中的缺陷已得到认可，并制定了一种克服它们的策略。现在，该系统需要该系统回答问题，以便更多的生化和生物学方法，并且由于需要更详细的信息来开发基于病毒的治疗剂，并且作为疱疹病毒tegumentation的模型系统，需要增加这种需求。和DNA包装的发展。 我们的研究设计从一系列的内部实验（AIM 1）中进行了有条不紊的进行，这些实验将确定在重组杆状病毒感染细胞中获得组装所需的特定HCMV衣壳和衣壳相关的蛋白质。作为这些研究的一部分，我们将研究HCMV衣壳需要通过表面结合蛋白（例如最小的衣壳蛋白（HCMV PUL48/49）或噬菌体“ CAP”蛋白（HCMV PUL7777）的假定对应物）进行表面结合蛋白稳定的可能性。 ，或者也许是紧密的衣壳相关的碱性磷蛋白（HCMV PUL32），在其他疱疹病毒中没有对应物。 我们还将为HCMV（sub-aim 1a）建立一个无细胞的体外衣壳组装系统，作为一种补充和替代的细胞组装方法。我们将使用为HSV组件开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，而且还可以证明最直接，最方便的方法来识别和克服对电池内方法的缺点。 最近的研究表明，嵌合CMV支架蛋白可以驱动HSV和KSHV衣壳壳的组装。在AIM 2中，将测试功能中断的突变在基本的HCMV UL80脚手架蛋白（PUL80.5和PUL80A）中的影响，以评估该方法的适用性，以探测capsID形成所涉及的分子相互作用。该系统使我们能够克服脚手架蛋白的基本本质以及无法研究HCMV感染细胞中的这些功能所施加的障碍。 该R21提案的结果将是开发用于高优先性人类病原体的体外包膜组装系统。该系统的未来开发，以结合体外DNA包装，带状膜的包裹和包裹，将对针对该病毒的新型和新型抗病毒毒策略的发展产生重大影响。 HCMV是感染人们的九种疱疹病毒之一 和三种人β-疱疹病毒的原型。它引起了显着的发病率和死亡率，尤其是与移植感染和免疫受损组有关。需要采用新方法来预防和治疗所有疱疹病毒，并且通过该项目开发的组装系统将有助于努力。 公共卫生相关性：巨细胞病毒（CMV）是感染人并引起重大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该病毒的感染，而开发此类药物的靶标的一种来源是病毒衣壳组装途径。用于研究CMV衣壳组件体外的模型系统将使这是可行且有吸引力的利用领域。我们建议根据经验建立这样的人类CMV系统，鼓励初步研究的结果，并从与Epstein-Barr病毒和Kaposi的肉瘤相关疱疹病毒的同样生产性系统中获得的新知识以及类似的生产性系统获得的新知识。 。