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: 7494608
• 负责人: Rebecca Ethel Taurog
• 金额: $ 4.96万
• 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 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7494608
• 关键词: Archaea; Biochemical; Buffers; Capsid Proteins; Condition; 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（申请人提供）：岛屿硫化叶菌棒状病毒（SIRV）是一种纤维状病毒，感染世界各地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病毒基因组周围的外壳蛋白组成;在病毒体的末端是一个塞子和三个尾纤维。已发现纯化的SIRV外壳蛋白在低pH下孵育时，在不存在DMA的情况下自组装成长的螺旋状病毒样颗粒（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物化学和结构基础将为极端环境中生存的要求提供新的见解。我建议表征SIRV外壳蛋白组装，并确定外壳蛋白的结构，通过X射线晶体学和冷冻电子显微镜（cryo-EM）的VLP。将详细研究外壳蛋白自组装成VLP的条件和该过程的动力学。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射线晶体学确定。由于纤维状病毒颗粒结晶的困难，VLP的结构将通过cryo-EM确定，使用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cryo-EM重建相结合，以创建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，这将成为VLP组装的进一步生化研究的基础。自组装病毒颗粒已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中空内部的能力。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、嗜酸性质，SIRV可用于需要高热或低pH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。