Structure and Function of Bacteriophage Proteins

感染细菌的病毒（称为噬菌体）必须能够识别并附着在特定的宿主细胞上，然后触发一种机制，将病毒基因组（其核酸）注入宿主细胞。病毒核酸被包装在一种叫做衣壳的蛋白质外壳内，并且大多数噬菌体在衣壳上装配有尾部，病毒核酸可以通过该尾部注射到宿主细胞中以感染宿主细胞。噬菌体（一个小的单链DNA噬菌体家族）在感染时而不是在初始噬菌体组装时产生它们的尾部。该项目旨在研究这一过程，并将其与另一种具有固定尾巴的噬菌体C1的结构和功能特性进行比较。噬菌体的分析提供了对极其复杂的分子机器的结构和功能的深入了解，这可以为生物技术的创新提供深入了解。该项目将为学生和博士后研究员提供生物物理学和生物学界面的跨学科培训。将二十面体phiX 174噬菌体的结构和功能与短尾病毒C1的结构和功能进行比较，使用X射线晶体学来研究病毒组分，使用冷冻电子显微镜和电子断层扫描来研究病毒入侵宿主细菌的过程。 该项目有两个具体目标。具体目的1：研究phiX 174基因组通过噬菌体编码的H管将其基因组转位到E. coli宿主细胞。a.将确定phiX 174病毒体中尾管的组装状态。组装的尾部由病毒编码的H蛋白的十个拷贝组成，形成卷曲螺旋结构。B.使用cryoEM和cryoTM测定噬菌体ST-1（phiX 174的近亲）与宿主细胞脂多糖分子之间的相互作用。ST-1需要用于这些实验，因为E. phiX 174的大肠杆菌宿主太厚，以至于电子束不能穿透而没有太多的衰减。C.通过改变A域和B域的相对长度以及通过改变H管的长度来研究H管组装。D.其他远亲微病毒科中H蛋白的结构有待确定。e. phiX 174和噬菌体T7的DNA导管中系统存在的Gln残基将被改变以确定它们的功能。F.分析H管和ssDNA之间的复合物。 具体目标2：确定噬菌体C1的结构。a.使用cryoEM确定C1头的近原子分辨率结构。B.使用X射线晶体学确定C1尾蛋白的结构。

A virus that infects bacteria (known as a bacteriophage) must be able to recognize and attach to the specific host cell and then trigger a mechanism by which the viral genome (its nucleic acid) is injected into the host cell. The viral nucleic acid is packaged inside a protein coat called the capsid, and the majority of bacteriophages are assembled with a tail on the capsid through which the viral nucleic acid can be injected into the host cell to infect it. It has recently become apparent that members of the Microviridae (a family of small single stranded DNA bacteriophages) generate their tail at the time of infection rather than at the time of initial phage assembly. This project is directed at investigating this process and comparing it with the structural and functional properties of bacteriophage C1, another type of bacteriophage that has a fixed tail. The analysis of bacteriophages provides insight into the structure and function of extraordinarily intricate molecular machines, which could provide insight for innovations in biotechnology. This project will provide interdisciplinary training to students and postdoctoral fellows at the interface of biophysics and biology. The structure and function of the small, icosahedral phiX174 bacteriophage is to be compared with that of the podovirus C1, using X-ray crystallography to study the viral components and cryo-electron microscopy as well as electron tomography to study the viruses as they invade their host bacteria. This project has two specific aims. Specific Aim 1: The mechanism by which the phiX174 genome translocates its genome through the phage encoded H tube into an E. coli host cell is to be determined. a. The state of assembly of the tail tube in phiX174 virions is to be determined. The assembled tail consists of ten copies of the virally encoded H protein that form a coiled coil structure. b. The interaction between phage ST-1 (a close relative of phiX174) and host cell lipopolysaccharide molecules is to be determined using cryoEM and cryoTM. ST-1 needs to be used for these experiments because the E. coli hosts for phiX174 are too thick to allow the electron beam to penetrate without too much attenuation. c. The H-tube assembly is to be studied, by changing the relative lengths of the A and B domains and by modifying the lengths of the H-tubes. d. The structures of the H protein in other, distantly related Microviridae are to be determined. e. The systematically occurring Gln residues in the DNA conduit of phiX174 and phage T7 are to be changed in order to establish their function. f. Complexes between the H tubes and ssDNA are to be analyzed. Specific Aim 2: The structure of bacteriophage C1 is to be determined. a. A near-atomic resolution structure of the C1 head is to be determined using cryoEM. b. The structure of the C1 tail proteins are to be determined using X-ray crystallography.