Structure and Function of Bacteriophage Proteins

Purdue University, West Lafayette IN

Investigators

Abstract

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.