Electron cryo-microscopy of phage P22

描述（由申请人提供）：病毒感染显著影响所有形式的生命。由于每种病毒和噬菌体都需要正确的衣壳蛋白（CP）结构，因此必须最终确定这些CP组装的机制，以设计有效的方法来规避感染或其传播。噬菌体P22在包括人类疱疹病毒在内的众多dsDNA病毒的主要CP中具有组装特征和高度的结构保守性，并将在拟议的研究中作为模型系统。我们将研究P22感染沙门氏菌的感染阶段，采用高压冷冻和冷冻置换（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P22附着到细胞和随后释放的次要蛋白质gp 16，gp 7和gp 20进入细胞。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那些阶段，如原衣壳组装和成熟。此外，我们建议采用电子冷冻显微镜对单个颗粒，以确定发生在主要衣壳蛋白的病毒粒子成熟的结构转变。将这些强大的技术添加到我的曲目中并掌握它们，将有助于我在顶级机构中获得教职。

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.