Electron cryo-microscopy of phage P22

噬菌体 P22 的电子冷冻显微镜

• 批准号: 7935228
• 负责人: Kristin N Parent
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935228
• 关键词: Address; Adenoviruses; Bacterial Proteins; Bacteriophage P22; Bacteriophages; Biochemical Genetics; Biological Models; Capsid; Capsid Proteins; Cells; Cryoelectron Microscopy; DNA Sequence Rearrangement; Defect; Double Stranded DNA Virus; Elements; Event; Faculty; Fluorescence Microscopy; Freeze Substitution; Freezing; Goals; Herpesviridae; Homologous Gene; Homology Modeling; Human; Image; Infection; Institution; Life; Life Cycle Stages; Maps; Methods; Microtomy; Minor; Modeling; Play; Positioning Attribute; Protein Subunits; Proteins; Research; Role; Salmonella; Sampling; Site-Directed Mutagenesis; Staging; Structure; Techniques; Variant; Viral; Virion; Virus; Virus Assembly; Virus Diseases; ds-DNA; electron tomography; image reconstruction; particle; pressure; reconstruction

DESCRIPTION (provided by applicant): Viral infection significantly impacts all forms of life. Since every virus and bacteriophage requires correct organization of its capsid proteins (CPs), the mechanisms by which these CPs assemble must ultimately be determined in order to devise efficacious methods to circumvent infection or its spread. Bacteriophage P22 shares features of assembly and a high degree of structural conservation among the major CPs of numerous dsDNA viruses, including the human herpesviruses, and will serve as a model system in the proposed research. We will study the stages of infection in P22-infected Salmonella, employing both electron tomography of samples treated by High Pressure Freezing and Freeze Substitution (HPF/FS) and fluorescence microscopy. Initially, our focus will be on visualizing early events of infection, including P22 attachment to cells and subsequent release of the minor proteins gp16, gp7, and gp20 into cells. Ultimately, these methods will help us characterize many stages of the virus life cycle, including those that occur during the end of the cycle such as procapsid assembly and maturation. In addition, we propose to employ electron cryo-microscopy on single particles to determine the structural transitions that occur in the major capsid protein as the virion matures. Adding and mastering these powerful techniques to my repertoire will bolster my path towards a faculty position at a top tier institution.

描述（由申请人提供）：病毒感染显着影响所有形式的生活。由于每种病毒和噬菌体都需要正确组织其衣壳蛋白（CPS），因此必须确定这些CPS组装的机制，以设计有效的方法来避免感染或扩散。噬菌体P22在许多DSDNA病毒（包括人疱疹病毒）的主要CPS中具有组装的特征和高度的结构保护，并将在拟议的研究中充当模型系统。我们将研究P22感染沙门氏菌的感染阶段，采用通过高压冻结和冷冻取代（HPF/FS）和荧光显微镜处理的样品的电子断层扫描。最初，我们的重点将是可视化感染的早期事件，包括对细胞的P22附着以及随后释放次要蛋白质GP16，GP7和GP20在细胞中。最终，这些方法将帮助我们表征病毒生命周期的许多阶段，包括在周期结束时发生的阶段，例如procapsid组装和成熟。此外，我们建议在单个颗粒上使用电子冷冻微观显微镜，以确定随着病毒素的成熟，主要衣壳蛋白中发生的结构过渡。在我的曲目中添加和掌握这些强大的技术将加强我在顶级机构的教职员工职位的道路。