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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM076661-09
Application #
8963999
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sakalian, Michael
Project Start
2007-04-15
Project End
2019-08-31
Budget Start
2015-09-01
Budget End
2016-08-31
Support Year
9
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
614209054
City
Storrs-Mansfield
State
CT
Country
United States
Zip Code
Asija, Kunica; Teschke, Carolyn M (2018) Lessons from bacteriophages part 2: A saga of scientific breakthroughs and prospects for their use in human health. PLoS Pathog 14:e1006970
Asija, Kunica; Teschke, Carolyn M (2018) Lessons from bacteriophages part 1: Deriving utility from protein structure, function, and evolution. PLoS Pathog 14:e1006971
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
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
Keifer, David Z; Motwani, Tina; Teschke, Carolyn M et al. (2016) Measurement of the accurate mass of a 50 MDa infectious virus. Rapid Commun Mass Spectrom 30:1957-62
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
Harprecht, Christina; Okifo, Oghenefejiro; Robbins, Kevin J et al. (2016) Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain. J Biol Chem 291:11359-72
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

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