This proposal addresses large-scale conformational changes associated with virus particle maturation and their effect on particle stability and infectivity. Biological assembly is a delicate process requiring subunit annealing and self-correction in the formation of the functional entity. Intracellular signaling events are adequately initiated with this level of stability, but virus particles have an extra-cellular and extra-organism portion of their life cycle that requires robust stability. Particle maturation provides a mechanism to accommodate weak interactions required for proper assembly (i.e. the provirion) with a chemical program, encoded in the provirion, that leads to maturation and stability. We will utilize systems developed during the last period of support to investigate the detailed chemistry of large scale conformational changes and their driving forces, the mechanisms of stabilizing quasi-equivalent capsid interactions, the activation of auto-catalytic chemistry and the onset of infectivity. The archeal, bacterial and eukaryotic virus systems chosen vividly illustrate the convergent evolution of this process with dramatically different pathways achieving closely similar final results. Our work has lead to novel applications of electron cryo-microscopy and image reconstruction (CryoEM) to study, in vitro, time resolved formation of auto-catalytic active sites during eukaryotic virus maturation, sub-nanometer asymmetric reconstructions of bacterial viruses and their maturation intermediates, and in vivo maturation of an archaeal virus related to the human adenovirus. Our focus during the next period of support will be the use of near-atomic resolution cryoEM to map the structures of the procapsid and mature particles of a virus that has an internal membrane, to use of electron cryo-tomography and maximum likelihood reconstructions to discern functionally important asymmetric features in near symmetric capsids, and to use hydrogen/deuterium exchange to increase the resolution of subunit chemical interactions in maturation intermediates of a eukaryotic virus. The results of this effort will identify vulnerable transitions during virus maturation and provide fundamental insights into large-scale conformational changes including their biophysical driving forces and chemistry.
This proposal extends our work on virus particle maturation;the process in which a virus transitions from a non-infectious provirion to an infectious virion. The process is virtually universal among animal viruses and is a worthy target for the development of antiviral agents as evidenced by the impact of HIV protease inhibitors. Our studies focus on archeal, bacterial, and eukaryotic viruses that we investigate in vivo and in vitro to establish the encoded programs that drive large-scale conformational changes in the provirions, which lead to their associated increases in particle stability and the gain of infectivity.
|McNulty, Reginald; Lokareddy, Ravi Kumar; Roy, Ankoor et al. (2015) Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22. J Mol Biol 427:3285-99|
|Routh, Andrew; Head, Steven R; Ordoukhanian, Phillip et al. (2015) ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3'-Azido cDNAs. J Mol Biol 427:2610-6|
|Routh, Andrew; Chang, Max W; Okulicz, Jason F et al. (2015) CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data. Methods 91:40-7|
|Campbell, Melody G; Kearney, Bradley M; Cheng, Anchi et al. (2014) Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200 kV. J Struct Biol 188:183-7|
|Tang, Jinghua; Kearney, Bradley M; Wang, Qiu et al. (2014) Dynamic and geometric analyses of Nudaurelia capensis Ï‰ virus maturation reveal the energy landscape of particle transitions. J Mol Recognit 27:230-7|
|Routh, Andrew; Johnson, John E (2014) Discovery of functional genomic motifs in viruses with ViReMa-a Virus Recombination Mapper-for analysis of next-generation sequencing data. Nucleic Acids Res 42:e11|
|Kearney, Bradley M; Johnson, John E (2014) Assembly and maturation of a T = 4 quasi-equivalent virus is guided by electrostatic and mechanical forces. Viruses 6:3348-62|
|Domitrovic, Tatiana; Movahed, Navid; Bothner, Brian et al. (2013) Virus assembly and maturation: auto-regulation through allosteric molecular switches. J Mol Biol 425:1488-96|
|Wang, Qiu; Matsui, Tsutomu; Domitrovic, Tatiana et al. (2013) Dynamics in cryo EM reconstructions visualized with maximum-likelihood derived variance maps. J Struct Biol 181:195-206|
|Johnson, John E (2013) Confessions of an icosahedral virus crystallographer. Microscopy (Oxf) 62:69-79|
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