A Structure-Function Analysis of the φX174 DNA Piloting Protein

Abstract

In order to initiate an infection, bacteriophages must deliver their large, hydrophilic genomes across their host’s hydrophobic cell wall. Bacteriophage φX174 accomplishes this task with a set of identical DNA piloting proteins. The structure of the piloting protein’s central domain was solved to 2.4 Å resolution. In it, ten proteins are oligomerized into an α-helical barrel, or tube, that is long enough to span the host’s cell wall and wide enough for the circular, ssDNA to pass through. This structure was used as a guide to explore the mechanics of φX174 genome delivery. In the first study, the H-tube’s highly repetitive primary and quaternary structure made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for the ability to perform the protein’s three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis. The tube’s inner surface was altered in the second study. The surface is primarily lined with amide and guanidinium containing amino acid side chains with the exception of four sites near the tube’s C-terminal end. The four sites are conserved across microvirus clades, suggesting that they may play an important role during genome delivery. To test this hypothesis and explore the general role of the amide and guanidinium containing side chains, the amino acids at these sites were changed to glutamine. The resulting mutants had a cold-sensitive phenotype at 22°C. Viral lifecycle steps were assayed in order to determine which step was disrupted by the mutant glutamine residues. The results support a model in which a balance of forces governs genome delivery: potential energy provided by the densely packaged viral genome and/or an osmotic gradient push the genome into the cell, while the tube’s inward facing residues exert a frictional force on the genome as it passes. Bacteriophage must first identify a susceptible host prior to genome delivery. In the final study, biochemical and genetic analyses were conducted with two closely related bacteriophages, α3 and ST-1. Despite ~90% amino acid identity, the natural host of α3 is Escherichia coli C, whereas ST-1 is a K-12-specific phage. To determine which structural proteins conferred host range specificity, chimeric virions were generated by individually interchanging the coat, spike, or DNA pilot proteins. Interchanging the coat protein switched host range. However, host range expansion could be conferred by single point mutations in the coat protein. The expansion phenotype was recessive: mutant progeny from co-infected cells did not display the phenotype. Novel virus propagation and selection protocols were developed to isolate host range expansion mutants. The resulting genetic and structural data were consistent enough that host range expansion could be predicted, broadening the classical definition of antireceptors to include interfaces between protein complexes within the capsid.