How does the KSHV small capsid protein function to promote self-assembly?

KSHV 小衣壳蛋白如何发挥促进自组装的作用？

• 批准号: 8570572
• 负责人: PRASHANT J DESAI
• 金额: $ 22.84万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-11 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8570572
• 关键词: Achievement; Address; Antiviral Agents; Bacteriophages; Baculoviruses; Binding; Biochemical; Biological; Biological Assay; Capsid; Capsid Proteins; Cell-Free System; Cells; Chimeric Proteins; Data; Defect; Development; Disease; Future; Genetic; Genome; Goals; Herpesviridae; Herpesvirus 1; Higher Order Chromatin Structure; Human Herpesvirus 4; Human Herpesvirus 8; In Vitro; Insecta; Kaposi Sarcoma; Lead; Location; Lytic; Mediating; Mediator of activation protein; Methods; Monitor; Multicentric Angiofollicular Lymphoid Hyperplasia; Nuclear; Oncogenic; Open Reading Frames; Outcome; Outcome Study; Pathway interactions; Peptide Hydrolases; Process; Production; Property; Protein Analysis; Protein Binding; Proteins; Protocols documentation; Recombinants; Role; Scaffolding Protein; Sedimentation process; Site; Specific qualifier value; Staging; Structure; System; Testing; Venus; Virus; Work; base; crosslink; gammaherpesvirus; inhibitor/antagonist; mutant; novel; primary effusion lymphoma; protein expression; protein function; public health relevance; research study; scaffold; self assembly; small molecule; tumor

DESCRIPTION (provided by applicant): Herpesvirus viruses encode six proteins that come together in a highly regulated and orchestrated fashion to form a protective coat around the virus genome. Virus and phage protein coats have been studied as paradigms for how proteins interact and self-assemble into higher order structures. This proposal is based on our studies of the Kaposi's sarcoma-associated herpesvirus (KSHV) small capsid protein (SCP) which is encoded by open reading frame (ORF) 65. The KSHV SCP is essential for assembly unlike those of other herpesviruses. Thus, we propose to study and elucidate how this protein promotes self-assembly of icosahedral capsids. Our working hypothesis is that the gammaherpesvirus SCP is an important mediator of stable capsid shell assembly. We propose an unconventional mechanism but one that phage capsids also use. Thus, because of its location on the hexons which are made up of the major capsid protein (MCP) and because it must function at a stage prior to the formation of visible (by ultrastructural methods) assemblies we propose that it acts as an external scaffold or cross-link that strengthens and stabilizes hexon formation. In addition, genetic data from mutants of ORF65 that cannot assemble suggests possibly a novel mechanism whereby binding of SCP to the MCP creates an allosteric change that strengthens and reinforces the MCP interaction with the internal scaffold protein. We propose experiments that will test this hypothesis. If proven correct this would change the view that the SCP is simply a capsid decoration protein. It would reveal an important role for this small protein at the initial steps in the assembly pathway. The Specific Aims proposed to achieve these goals are: Specific Aim 1. Discover the interactions and properties of the KSHV SCP (ORF65). I. Nuclear assembly site localization. II. Interactions of ORF65. III. Dynamic functional state of ORF65. Specific Aim 2. Use a cell-free system and in vitro methods to discover the novel functions of the SCP. I. Cell-free assembly. II. Sedimentation analysis of sub-assemblies. III. In vitro MCP-scaffold protein binding assay.

描述（由申请人提供）：疱疹病毒编码六种蛋白质，这些蛋白质以高度调控和精心策划的方式聚集在一起，在病毒基因组周围形成保护层。病毒和噬菌体蛋白外壳已被作为蛋白质如何相互作用和自组装成更高阶结构的范例进行研究。该提案基于我们对卡波西肉瘤相关疱疹病毒 (KSHV) 小衣壳蛋白 (SCP) 的研究，该蛋白由开放阅读框 (ORF) 65 编码。与其他疱疹病毒不同，KSHV SCP 对于组装至关重要。因此，我们建议研究和阐明这种蛋白质如何促进二十面体衣壳的自组装。我们的工作假设是，伽马疱疹病毒 SCP 是稳定衣壳壳组装的重要介质。我们提出了一种非常规机制，但噬菌体衣壳也使用这种机制。因此，由于它位于由主要衣壳蛋白（MCP）组成的六邻体上，并且因为它必须在形成可见（通过超微结构方法）组件之前的阶段发挥作用，我们建议它充当外部支架或加强和稳定六邻体形成的交联。此外，无法组装的 ORF65 突变体的遗传数据表明可能存在一种新机制，即 SCP 与 MCP 的结合产生变构变化，从而增强 MCP 与内部支架蛋白的相互作用。我们提出实验来检验这一假设。如果被证明是正确的，这将改变 SCP 只是一种衣壳装饰蛋白的观点。它将揭示这方面的重要作用 在组装途径的初始步骤中的小蛋白质。为实现这些目标而提出的具体目标是： 具体目标 1. 发现 KSHV SCP (ORF65) 的相互作用和特性。一、核组装场地本地化。二. ORF65 的相互作用。三． ORF65 的动态功能状态。具体目标2.利用无细胞系统和体外方法发现SCP的新功能。 I.无细胞组装。二.子组件的沉降分析。三．体外 MCP 支架蛋白结合测定。