Phages as Lenses to Study Virus Assembly and Virus: Host Interactions

SUMMARY Icosahedral capsid assembly is a highly coordinated process involving sequential addition of multiple proteins, ultimately leading to an infectious virion of proper size and morphology. One long-term goal for this project is to achieve a mechanistic understanding of the protein:protein interactions involved in capsid assembly. The development of new anti-viral drugs is impeded by a lack of understanding of how viral capsid proteins are programmed to adopt the correct conformations to produce the correct assembly product. Capsid assembly will be investigated using bacteriophage P22, a model dsDNA virus. In phage P22, herpesvirus and many other dsDNA viruses, the capsid is formed from a coat protein having the ubiquitous HK97 fold. The initial assembly product is a procapsid (PC). Scaffolding protein (SP) directs proper assembly of coat protein (CP) to form PCs. SP also directs the incorporation of the portal protein complex, which is essential for genome encapsidation. Phage P22 provides an excellent model assembly system because complex in vivo processes are easily mimicked in vitro. The simple genetics and well-established biochemistry of phage P22 offers significant advantages as an assembly model over complex mammalian dsDNA viruses. Our goals for this project are to 1) reveal how different regions of SP interact with all of the capsid proteins to control assembly and yield of PCs with the correct morphology, 2) understand the SP structure and arrangement inside of PCs, and 3) understand the signal relayed by portal protein during DNA packaging that lead to SP exit and the virus capsid maturation. Another goal is to understand understudied systems involved in P22’s interaction with host cells and proteins. We will determine the mechanism of superinfection exclusion by P22’s SieA, and the function of P22’s gp14. 1) SieA prevents the delivery of DNA across the Salmonella inner membrane by P22 and P22-like phages through interaction with their ejection proteins, which are used to make the genome delivery tube. But how SieA does this is not understood. 2) P22’s gp14, which is encoded in the structural gene cluster, has no known function. We have shown that it is a spermidine acetyl transferase, by why P22 codes for this enzyme is a mystery. A combination of molecular biology, genetic and -omics approaches along with biochemistry and structural analyses of proteins and particles will be used in the investigation of the assembly process and virus:host interactions.