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）：疱疹病毒病毒编码六种以高度调节和精心策划的方式融合在一起的蛋白质，以形成病毒基因组周围的保护外套。已经研究了病毒和噬菌体蛋白涂层，作为蛋白质如何相互作用并自组装成高阶结构的范例。该提案基于我们对卡波西肉瘤相关的疱疹病毒（KSHV）小皮环蛋白（SCP）的研究，该蛋白质（SCP）由开放式阅读框架（ORF）65编码。KSHVSCP对组装至关重要，对于与其他疱疹病毒不同。因此，我们建议研究和阐明该蛋白质如何促进二十面体帽的自组装。我们的工作假设是，伽马梅犬SCP是稳定的衣壳壳组件的重要介体。我们提出了一种非常规的机制，但噬菌体包包也使用。因此，由于其位于由主要的衣壳蛋白（MCP）组成的六角形上，并且由于它必须在形成可见的阶段（通过超微结构方法）组件之前在阶段起作用，因此我们建议它充当外部支架或交叉链接，以增强和稳定己孔的形成。此外，无法组装的ORF65突变体的遗传数据可能表明了一种新型机制，即SCP与MCP结合会产生变构变化，从而增强并增强MCP与内部支架蛋白的相互作用。我们提出的实验将检验这一假设。如果被证明是正确的，这将改变SCP仅仅是帽固定装饰蛋白的观点。这将揭示这一重要作用 小蛋白在组装途径的初始步骤处。提出实现这些目标的具体目的是：特定目标1。发现KSHV SCP（ORF65）的相互作用和特性。 I.核装配部位定位。 ii。 ORF65的相互作用。 iii。 ORF65的动态功能状态。特定目标2。使用无细胞系统和体外方法发现SCP的新功能。 I.无细胞组件。 ii。子组件的沉积分析。 iii。体外MCP-SCASCODLOLD蛋白结合测定法。