Despite many years of research, a comprehensive understanding of the process of viral capsid assembly at the molecular level has not yet been developed. The long-term goal of this project is to understand the mechanistic details of assembly of icosahedral viruses, which can then be used as the basis for development of antivirals targeted at capsid assembly. We propose to investigate assembly of the dsDNA bacteriophage P22, which provides an excellent model for icosahedral virus assembly. Our specific hypothesis is that viral capsid assembly is driven by multiple specific weak protein:protein interactions of the subunits during assembly. Phage P22 first assembles a procapsid into which dsDNA is packaged. In vitro procapsid-like particles can be assembled simply by mixing together coat and scaffolding proteins in the appropriate conditions. The proposed work combines rigorous thermodynamic analysis of assembly with biochemical and genetic approaches. We propose to first characterize the thermodynamics of P22 procapsid assembly to determine how ionic interactions, and entropic and enthalpic forces are involved in correct assembly of capsids. The role of scaffolding protein in proper assembly will be described, also by determining thermodynamic values for the assembly reaction by using scaffolding protein variants. The controlled of addition of capsid subunits during elongation will be characterized through equilibrium analysis of the association of subunits with partial capsids. Secondly, the sites and nature of the interaction between coat and scaffolding protein will be determined by a combination of molecular biology, phage genetics and biochemical techniques. Lastly, how scaffolding protein is organized within procapsids will be established through techniques using electron microscopy. The research proposed is relevant to public health because thoroughly characterizing capsid assembly will allow the step(s) that are the best targets for anti-viral drugs to be identified. In addition, these studies will highlight the important interactions between capsid subunits, which are required for proper assembly of viruses.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM076661-04
Application #
7795199
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Flicker, Paula F
Project Start
2007-04-15
Project End
2011-09-29
Budget Start
2010-04-01
Budget End
2011-09-29
Support Year
4
Fiscal Year
2010
Total Cost
$272,646
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
06269
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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