Year entered the Ph.D. Program: Fall 2012
Research Advisor: Bentley Fane
Research Topic: Bacteriophage Genome Entry
Lab phone number: 621-5839
To initiate an infection, bacteriophage ΦX174 uses a collection of identical DNA piloting proteins to move its genome into a host Escherichia coli. The crystal structure of the piloting protein’s central domain was solved to 2.4 Å resolution (Sun et al, Nature 2014). In the structure, the piloting proteins are seen oligomerized into a decameric tube that is long enough to span the E. coli membranes and wide enough for the circular, ssDNA genome to pass through. The inner surface of the tube is primarily lined with amide and guanidinium containing amino acid side chains, with such side chains occupying 18 of the 23 inward facing sites seen in the crystal structure. As glutamine, asparagine, and arginine side chains can interact with DNA nucleotides, we hypothesize that these inward facing residues may be generating hydrodynamic drag on the passing viral genome, thereby regulating viral genome release. Regulation may be necessary as the capsid is highly pressurized with the densely packaged genome. Releasing this pressure in small amounts may prevent tube failure or DNA tangling. To test this hypothesis, we have mutated some of the inward facing, non-amide and non-guanidinium residues to glutamine. The resulting mutants had a cold-sensitive phenotype and could no longer form plaques at 22°C. All steps of the viral lifecycle involving the piloting protein were assayed at 22°C in order to determine which step was inhibited by the extra glutamines. Host adsorption, infection initiation, and virion assembly were not affected by the mutations. Genome delivery, however, appeared to be inhibited at 22°C. The genome delivery step was dissected into two parts: 1) channel formation was observed by monitoring the efflux of intra-cellular K+, and 2) the location of the infecting genomes in the host membranes was tracked with qPCR following membrane isolation and separation. Preliminary results suggest that genome delivery channels do open, however a large proportion of the infecting genomes do not reach the host’s cytoplasmic membrane, the site of stage 1 viral DNA synthesis. This suggests that the extra glutamine residues may be increasing the amount of hydrodynamic drag on the passing viral genome. At lower temperatures, the pressure in the capsid may be reduced, and with increased drag the infecting genome may become kinetically trapped within the genome delivery tube.