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控和精心编排的方式聚集在一起，在病毒基因组周围形成一层保护膜。病毒和噬菌体蛋白外壳被研究为蛋白质相互作用和自组装成高阶结构的范例。这一建议是基于我们对开放阅读框（ORF） 65编码的卡波西肉瘤相关疱疹病毒（KSHV）小衣壳蛋白（SCP）的研究。与其他疱疹病毒不同的是，KSHV SCP对于组装是必不可少的。因此，我们建议研究和阐明该蛋白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是稳定衣壳组装的重要介质。我们提出了一种非常规的机制，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）组成的六方体上，并且由于它必须在可见（通过超微结构方法）组装形成之前的阶段起作用，我们建议它作为外部支架或交联，加强和稳定六方体的形成。此外，来自无法组装的ORF65突变体的遗传数据表明，SCP与MCP结合可能会产生一种新的机制，即变构变化，从而加强和加强MCP与内部支架蛋白的相互作用。我们提出了一些实验来验证这一假设。如果被证明是正确的，这将改变SCP仅仅是一种衣壳装饰蛋白的观点。它将揭示这方面的重要作用

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.