During the past three years of NSF support, progress was made in understanding the regulation of shape and size in virus assembly using bacteriophage T4. Studies on head length regulation have shown that the DNA inside the virus is selected for packaging at specific lengths that can be interpreted by reference to icosahedral surface lattice theory. DNA packaging in T4 is therefore dependent on the design principles used by the capsid in setting its length parameters. Use of pulsed-field electrophoresis of DNA has provided the highest resolution yet for the separation of T4 DNA molecules. The new work proposed here will focus on how the measuring system that determines DNA packaging operates. The head of phage T4 is elongated, not icosahedral as for most viruses. Mutations in the internal protein core or scaffold proteins all cause the head to revert to its icosahedral form. All mutations that caused elongated heads (called giants) are located in the gene coding for the virus coat protein (gp 23). We will test the idea that gp 23 alone controls head elongation using cloned genes for both the scaffold and the coat protein in expression vectors, site-directed deletions and mutagenesis, and molecular structure determination. Purified gp23 will be subjected to crystallization conditions for x-ray structure determination. Of the four models for T4 head length determination that have been proposed, it is believed two can be eliminated (the simple template and the kinetic models). This will now extend and confirm preliminary data that suggest that length is controlled by a combination of a two-component protein vernier mechanism and stabilization of accumulated strain at the icosahedral vertices. Together, the proposed experiments will help substantially in understanding of the mechanisms of shape and size control, and DNA packaging, at the subcellular level.
