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

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节和协调的方式聚集在一起，在病毒基因组周围形成保护性外壳。病毒和噬菌体蛋白质外壳已被研究作为蛋白质如何相互作用和自组装成更高级结构的范例。这个建议是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架（ORF）65编码。与其他疱疹病毒不同的是，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，γ疱疹病毒SCP是一个重要的调解人的稳定衣壳壳组装。我们提出了一个非传统的机制，但噬菌体衣壳也使用。因此，由于其在由主要衣壳蛋白（MCP）组成的六邻体上的位置，并且由于其必须在形成可见（通过超微结构方法）组件之前的阶段起作用，我们建议其充当外部支架或交联，以加强和稳定六邻体形成。此外，来自不能组装的ORF 65突变体的遗传数据表明可能存在一种新的机制，即SCP与MCP的结合产生变构变化，从而加强和强化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。这将揭示出一个重要的作用， 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。探索KSHV SCP（ORF 65）的相互作用和特性。I.核组装地点定位。二. ORF 65的相互作用。三. ORF 65的动态功能状态。具体目标2。使用无细胞系统和体外方法来发现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。

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.