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其衣壳蛋白（CPs）的正确组织，因此必须最终确定这些CPs组装的机制，以便设计有效的方法来避免感染或传播。噬菌体P22在包括人类疱疹病毒在内的许多dsDNA病毒的主要cp中具有组装和高度结构保守的特征，将作为拟研究的模型系统。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替代（HPF/FS）处理的样品的电子断层扫描和荧光显微镜。最初，我们的重点将是可视化感染的早期事件，包括P22附着在细胞上以及随后将次要蛋白gp16、gp7和gp20释放到细胞中。最终，这些方法将帮助我们描述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，我们建议在单个颗粒上使用电子冷冻显微镜来确定随着病毒粒子成熟，主要衣壳蛋白中发生的结构转变。将这些强大的技术添加并掌握到我的曲目中，将有助于我在顶级机构担任教职。

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.