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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Motwani, Tina; Lokareddy, Ravi K; Dunbar, Carmen A et al. (2017) A viral scaffolding protein triggers portal ring oligomerization and incorporation during procapsid assembly. Sci Adv 3:e1700423
Tripler, Therese N; Teschke, Carolyn M; Alexandrescu, Andrei T (2017) NMR assignments for the insertion domain of bacteriophage Sf6 coat protein. Biomol NMR Assign 11:35-38
Lokareddy, Ravi K; Sankhala, Rajeshwer S; Roy, Ankoor et al. (2017) Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation. Nat Commun 8:14310
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Keifer, David Z; Motwani, Tina; Teschke, Carolyn M et al. (2016) Acquiring Structural Information on Virus Particles with Charge Detection Mass Spectrometry. J Am Soc Mass Spectrom 27:1028-36
Wu, Weimin; Leavitt, Justin C; Cheng, Naiqian et al. (2016) Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging. MBio 7:
D'Lima, Nadia G; Teschke, Carolyn M (2015) A Molecular Staple: D-Loops in the I Domain of Bacteriophage P22 Coat Protein Make Important Intercapsomer Contacts Required for Procapsid Assembly. J Virol 89:10569-79
Suhanovsky, Margaret M; Teschke, Carolyn M (2015) Nature's favorite building block: Deciphering folding and capsid assembly of proteins with the HK97-fold. Virology 479-480:487-97
Tripler, Therese N; Maciejewski, Mark W; Teschke, Carolyn M et al. (2015) NMR assignments for the insertion domain of bacteriophage CUS-3 coat protein. Biomol NMR Assign 9:333-6

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