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. The 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 central hypothesis is viral capsid assembly is driven by specific weak protein: protein interactions, which control nucleation and elongation to form the proper assembly products. In this granting period we will test our central hypothesis with the following aims.
Aim 1. Define how communication between domains of coat protein affects capsid morphology. Our data suggest that proper capsid morphology is controlled by communication between domains of CP, thereby affecting the curvature of the subunit. We will test this hypothesis by generating and characterizing site- directed mutants in different domains of P22 CP.
Aim 2. Determine the structure and function of the I-domain from distantly related phages. We defined important roles for an inserted domain in the folding and assembly of P22 coat protein. Phages Sf6 and CUS-3 have low coat protein sequence identity but have an identifiable inserted domain. We will determine the NMR solution structure, and the function of the inserted domain in the CP folding and assembly from these related phages.
Aim 3. Elucidate the protein conformational changes occurring during assembly. Protein conformational changes regulate proper capsid assembly. We will use single molecule FRET experiments to understand how individual capsid proteins in a population change conformations between monomeric and assembled states.
Aim 4. Investigate the assembly of the portal protein complex during PC assembly. Portal protein is essential for genome packaging, yet how this complex is assembled into procapsids at a single vertex is not understood. Incorporation of portal protein will be characterized using RNA atpamers identified by SELEX.
All viruses must self-assemble, a process which is controlled by the conformation and interactions of viral proteins. The proposed research is relevant to public health because a deeper understanding of the protein interactions that drive virus assembly will aid in development of novel anti-viral therapeutics targeted at virus assembly. Bacteriophage P22 provides a simple model system for complex dsDNA viruses like herpesviruses. Therefore, bacteriophage P22 will be used in a detailed analysis of the protein interactions required for assembly dsDNA viruses.
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