Biochemical and structural characterization of self-assembling viral particles

描述（由申请人提供）：岛硫虫棒状病毒（SIRV）是一种纤维状病毒，感染在世界各地的热喷口和温泉中发现的嗜热、嗜酸古菌。该病毒由包裹在线性dsDNA病毒基因组上的外壳蛋白组成；在病毒粒子的末端有一个塞和三条尾纤维。纯化的SIRV外壳蛋白在没有DMA的低pH条件下自组装成长螺旋状病毒样颗粒（VLPs）。这种组装是可逆的，通过将VLPs重新引入高pH缓冲液中可以获得单体外壳蛋白。pH驱动病毒颗粒组装的生化和结构基础将为极端环境下的生存需求提供新的见解。我建议表征SIRV外壳蛋白组装，并通过x射线晶体学和低温电子显微镜（cryo-EM）确定外壳蛋白的结构。本文将详细研究外壳蛋白自组装成VLPs的条件和这一过程的动力学。为了研究dsDNA在病毒组装中的作用，还将研究dsDNA存在下VLP的形成。单分子SIRV外壳蛋白的结构将由x射线晶体学测定。由于纤维状病毒颗粒难以结晶，VLP的结构将通过冷冻电镜确定，利用VLP的螺旋对称性来帮助重建三维结构。然后将涂层蛋白的x射线晶体结构与VLP的低温电镜重建相结合，创建VLP的准原子模型。该模型将用于识别驱动病毒组装的分子间相互作用，这将形成VLP组装的进一步生化研究的基础。自组装病毒颗粒已经成为许多制药和纳米技术应用的目标，因为它们具有规则的结构和将小分子包装在其中空内部的能力。SIRV VLPs是纤维状的，就像烟草花叶病毒一样，它被用作合成纳米线的平台。由于其嗜热、嗜酸的性质，SIRV可能在需要高温或低ph的纳米技术应用中很有用。此外，SIRV VLP组装的可逆性可能被用作在VLP内将治疗药物或显像剂输送到特定器官的机制。

DESCRIPTION (provided by applicant): The Sulfolobus islandicus rod-shaped virus (SIRV) is a fibrous virus that infects thermophilic, acidophilic Archaea found in thermal vents and hot springs around the world. The virus is composed of a coat protein wrapped around the linear dsDNA viral genome; at the termini of the virion are a plug and three tail fibers. The purified SIRV coat protein has been found to self-assemble into long, helical virus-like particles (VLPs) when incubated at low pH, in the absence of DMA. This assembly is reversible, and monomeric coat protein can be obtained by reintroducing VLPs into high pH buffer. The biochemical and structural bases of the pH- driven assembly of the virus particle will provide new insights into the requirements for survival in extreme environments. I propose to characterize the SIRV coat protein assembly and determine the structure of both the coat protein by X-ray crystallography and the VLP by cryoelectron microscopy (cryo-EM). The conditions under which the coat protein self-assembles into VLPs and the kinetics of this process will be investigated in detail. To investigate the role of dsDNA in viral assembly, the formation of the VLP in the presence of dsDNA will also be studied. The structure of the monomeric SIRV coat protein will be determined by X-ray crystallography. Due to the difficulty of crystallizing fibrous virus particles, the structure of the VLP will be determined by cryo-EM, using the helical symmetry of the VLP to aid in the reconstruction of the three dimensional structure. The X-ray crystal structure of the coat protein will then be combined with the cryo- EM reconstruction of the VLP to create a quasi-atomic model of the VLP. This model will be used to identify the intermolecular interactions that drive viral assembly, which will form the basis of further biochemical studies of VLP assembly. Self-assembling viral particles have become a target for many pharmaceutical and nanotechnology applications, due to their regular structure and the ability to package small molecules in their hollow interiors. The SIRV VLPs are fibrous like the tobacco mosaic virus, which has been used as a platform for synthesis of nanowires. Due to its thermophilic, acidophilic nature, SIRV may be useful in nanotechnology applications that require high heat or low pH. In addition, the reversibility of the SIRV VLP assembly may be utilized as a mechanism for delivering therapeutics or imaging agents inside the VLP to a specific organ.