Biochemical and structural characterization of self-assembling viral particles

自组装病毒颗粒的生化和结构表征

• 批准号: 8039521
• 负责人: Rebecca Ethel Taurog
• 金额: $ 2.61万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8039521
• 关键词: Archaea; Biochemical; Buffers; Capsid Proteins; Cryoelectron Microscopy; DNA; Data; Environment; Fiber; Fluorescence; Heating; Hot Springs; Image; Incubated; Ionic Strengths; Kinetics; Modeling; Nanotechnology; Nature; Organ; Pharmacologic Substance; Process; Property; Proteins; Roentgen Rays; Role; Structure; Sulfolobus islandicus Rod-Shaped Viruses; Tail; Therapeutic; Tobacco Mosaic Virus; Vent; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus-like particle; X-Ray Crystallography; base; density; image reconstruction; insight; intermolecular interaction; light scattering; monomer; particle; protein protein interaction; protein structure; reconstruction; small molecule; three dimensional structure; viral DNA

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.

描述（由申请人提供）：岛硫化叶菌杆状病毒（SIRV）是一种纤维状病毒，可感染世界各地热喷口和温泉中发现的嗜热、嗜酸古细菌。该病毒由包裹在线性 dsDNA 病毒基因组周围的外壳蛋白组成；病毒粒子的末端有一个插头和三个尾纤维。研究发现，在低 pH 值、没有 DMA 的情况下，纯化的 SIRV 外壳蛋白会自组装成长螺旋状病毒样颗粒 (VLP)。这种组装是可逆的，并且可以通过将VLP重新引入高pH缓冲液中来获得单体外壳蛋白。 pH驱动的病毒颗粒组装的生化和结构基础将为极端环境下的生存要求提供新的见解。我建议表征 SIRV 外壳蛋白组装，并通过 X 射线晶体学确定外壳蛋白的结构，并通过冷冻电子显微镜 (cryo-EM) 确定 VLP 的结构。将详细研究外壳蛋白自组装成 VLP 的条件以及该过程的动力学。为了研究 d​​sDNA 在病毒组装中的作用，还将研究 dsDNA 存在下 VLP 的形成。单体 SIRV 外壳蛋白的结构将通过 X 射线晶体学确定。由于纤维状病毒颗粒结晶困难，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，外壳蛋白的 X 射线晶体结构将与 VLP 的冷冻电镜重建相结合，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，这将构成 VLP 组装进一步生化研究的基础。自组装病毒颗粒由于其规则的结构和在中空内部包装小分子的能力，已成为许多制药和纳米技术应用的目标。 SIRV VLP 与烟草花叶病毒一样呈纤维状，已被用作合成纳米线的平台。由于其嗜热、嗜酸的性质，SIRV 可用于需要高热或低 pH 值的纳米技术应用。此外，SIRV VLP 组装的可逆性可用作将 VLP 内的治疗剂或显像剂递送至特定器官的机制。