Icosahedral viral capsid assembly is a highly coordinated process that involves addition of multiple protein subunits, ultimately leading to an infectious virion of proper size and morphology. Often identical coat protein subunits occupy non-identical (hexons and pentons) sites in the icosahedron, which is known as conformational switching. For many dsDNA viruses, scaffolding proteins are used to direct proper assembly of coat protein so that the hexons and pentons are arranged correctly. How capsid proteins are programmed to adopt the correct conformations such that the appropriate assembly product is formed is not understood in detail for any virus, and is the rationale for this project. In addition, the assembly process presents a viable therapeutic target because the repeated use of the same subunits means that a molecule that interferes with capsid subunit associations will be a particularly efficacious inhibitor. Thus, the long-term goal for this work is to achieve a mechanistic understanding of protein:protein interactions involved in capsid assembly and to concisely define how those interactions are employed in each step in proper assembly. Capsid assembly will be investigated using bacteriophage P22 as a model dsDNA virus. In phage P22, herpesvirus and many other dsDNA viruses, the initial product of assembly is a precursor capsid, known as the procapsid (PC). Scaffolding protein directs proper assembly of coat protein, the major capsid protein, to form PCs. Scaffolding protein also directs the incorporation of the portal protein complex in vivo. P22 assembly is an excellent model system because complex in vivo processes can be mimicked in vitro. When P22 purified coat and scaffolding protein monomers are mixed together, procapsid-like particles are robustly generated. The simple genetics and well established biochemistry of P22 offers significant advantages as an assembly model over complex eukaryotic dsDNA viruses. The central hypothesis for this project is that capsid assembly is driven by specific weak protein:protein interactions, and is finely tuned by these interactions during nucleation and elongation to form the proper assembly products. The objective for this granting period is to test our central hypothesis through a detailed analysis of the protein:protein interactions that drive proper P22 procapsid assembly by pursuing the following three specific aims: 1) Identify regions in domains of coat protein that are involved in virus form determination;2) Elucidate the role of the telokin domain in P22 capsid assembly and stabilization;3) Understand scaffolding protein control of P22 capsid assembly. Each of these aims is supported by significant preliminary data generated in the investigator's and colaborators'labs. This project is innovative because the well established biochemical and genetic assays of the P22 system will be combined with recent structural data and used to interrogate the role of the capsid protein interactions in virion assembly. The proposed research is significant because the outcome will be a detailed mechanistic understanding of virion assembly due to weak interactions of capsid proteins.

Public Health Relevance

The proposed research is relevant to public health because deeper, and generalizable, understanding of the protein interactions that drive virus assembly will help with development of novel anti-viral therapeutics. Bacteriophage P22 is a simple model system for complex dsDNA viruses like Herpes viruses. Therefore, bacteriophage P22 will be used to in a detailed analysis of the protein interactions required for assembly dsDNA viruses.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-GGG-T (02))
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Sakalian, Michael
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University of Connecticut
Schools of Arts and Sciences
United States
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Rizzo, Alessandro A; Suhanovsky, Margaret M; Baker, Matthew L et al. (2014) Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling. Structure 22:830-41
Cortines, Juliana R; Motwani, Tina; Vyas, Aashay A et al. (2014) Highly specific salt bridges govern bacteriophage P22 icosahedral capsid assembly: identification of the site in coat protein responsible for interaction with scaffolding protein. J Virol 88:5287-97
Zlotnick, Adam; Suhanovsky, Margaret M; Teschke, Carolyn M (2012) The energetic contributions of scaffolding and coat proteins to the assembly of bacteriophage procapsids. Virology 428:64-9
Suhanovsky, Margaret M; Teschke, Carolyn M (2011) Bacteriophage P22 capsid size determination: roles for the coat protein telokin-like domain and the scaffolding protein amino-terminus. Virology 417:418-29
Cortines, Juliana R; Weigele, Peter R; Gilcrease, Eddie B et al. (2011) Decoding bacteriophage P22 assembly: identification of two charged residues in scaffolding protein responsible for coat protein interaction. Virology 421:1-11
Padilla-Meier, G Pauline; Teschke, Carolyn M (2011) Conformational changes in bacteriophage P22 scaffolding protein induced by interaction with coat protein. J Mol Biol 410:226-40
Parent, Kristin N; Khayat, Reza; Tu, Long H et al. (2010) P22 coat protein structures reveal a novel mechanism for capsid maturation: stability without auxiliary proteins or chemical crosslinks. Structure 18:390-401
Parent, Kristin N; Sinkovits, Robert S; Suhanovsky, Margaret M et al. (2010) Cryo-reconstructions of P22 polyheads suggest that phage assembly is nucleated by trimeric interactions among coat proteins. Phys Biol 7:045004
Suhanovsky, Margaret M; Parent, Kristin N; Dunn, Sarah E et al. (2010) Determinants of bacteriophage P22 polyhead formation: the role of coat protein flexibility in conformational switching. Mol Microbiol 77:1568-82
Teschke, Carolyn M; Parent, Kristin N (2010) 'Let the phage do the work': using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants. Virology 401:119-30

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