Biochemical and structural characterization of self-assembling viral particles

描述(申请人提供)：Sulfolobus islandicus杆状病毒(SIRV)是一种纤维状病毒，感染世界各地温泉和温泉中发现的嗜热、嗜酸古生菌。该病毒由包裹在线状dsDNA病毒基因组周围的外壳蛋白组成；在病毒粒子的末端有一个插头和三个尾部纤维。纯化的SIRV外壳蛋白在没有DMA的情况下，在低pH下孵育时，被发现能自组装成长的螺旋病毒样颗粒(VLP)。这种组装是可逆的，通过将VLP重新引入高pH缓冲液中可以获得单体外壳蛋白。PH驱动的病毒颗粒组装的生化和结构基础将为在极端环境中生存的要求提供新的见解。我建议对SIRV外壳蛋白组件进行表征，并通过X射线结晶学和冷冻电子显微镜(Cryo-EM)确定外壳蛋白和VLP的结构。将详细研究外壳蛋白自组装成VLP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，还将研究在dsDNA存在下VLP的形成。单体SIRV外壳蛋白的结构将通过X射线结晶学确定。由于纤维状病毒颗粒很难结晶，VLP的结构将由低温EM确定，利用VLP的螺旋对称性来帮助重建三维结构。然后，将外壳蛋白的X射线晶体结构与VLP的冷冻-EM重建相结合，创建VLP的准原子模型。这一模型将被用来确定驱动病毒组装的分子间相互作用，这将为进一步的VLP组装的生化研究奠定基础。自组装病毒颗粒由于其规则的结构和将小分子包裹在其中空内部的能力，已成为许多制药和纳米技术应用的目标。SIRV VLP是纤维状的，就像烟草花叶病毒一样，它已经被用作合成纳米线的平台。由于其嗜热、嗜酸的性质，SIRV可能在需要高热量或低pH的纳米技术应用中有用。此外，SIRV VLP组件的可逆性可用作将VLP内的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.