Biophysical, Biochemical, and Genetic Analysis A key step in the assembly of many viruses, including herpesviruses and poxviruses that cause significant morbidity and mortality in the human population, is the packaging of dsDNA into pre-assembled procapsids by an ATP-driven motor complex. Viral terminases comprise a major class of these packaging motors and carry out multiple functions, including binding and cleavage of DNA to initiate packaging of a genome-length of DNA from a concatemeric substrate, translocation of the DNA into the procapsid, and arrest and DNA cleavage to terminate the packaging reaction. We propose integrated genetic, biochemical, and biophysical studies to elucidate detailed mechanisms of the phage ? terminase packaging motor, a powerful model system for investigating general principles. Genetic methods are designed to identify mutants with altered packaging activities and determine phenotypic defects in vivo. Biochemical and kinetic studies are designed to interrogate packaging kinetics and assembly of viruses in vitro with defined sets of purified proteins. Biophysical analysis using optical tweezers enables detailed measurements of the packaging of single DNA molecules in real time. Each approach is designed to complement and support the others. The studies will focus on: (1) Identification of amino acid residues directly involved in motor function via detailed studies of the effect of mutations on motor subunit assembly, packaging efficiency and kinetics, ATP consumption, and infectious viral assembly;(2) A mechanistic dissection of the translocating motor to define DNA translocation rate, motor force generation, translocation step size and stepping dynamics, and coordination of motor subunits;(3) Interrogation of packaging termination and genome end maturation to define the physiokinetic factors that mediate sensing of the extent of packaging and motor arrest and DNA cleavage. The proposed studies will utilize a diverse scientific toolbox and build on solid preliminary studies that establish the genetic, biochemical, and biophysical framework used to dissect motor function. These studies will provide an unprecedented understanding of mechanochemical coupling (energy transduction) in the viral packaging motor and will yield mechanistic insight into key steps in virus assembly. The results will guide future studies on other virus systems and help to define general principles of ATP-driven molecular motors relevant to understanding homologous cellular complexes including RNA helicases and chromosome segregation factors.

Public Health Relevance

Our research is aimed at understanding viral DNA packaging, a key step in the assembly of many viruses, including herpesviruses and poxviruses that cause significant morbidity and mortality in the human population. Our studies of the basic principles of virus assembly will lead to a better understanding of this complex biological process and aid in the development of novel antiviral therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM088186-04
Application #
8653579
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sakalian, Michael
Project Start
2011-05-01
Project End
2015-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92093
delToro, Damian; Ortiz, David; Ordyan, Mariam et al. (2016) Walker-A Motif Acts to Coordinate ATP Hydrolysis with Motor Output in Viral DNA Packaging. J Mol Biol 428:2709-29
Keller, Nicholas; Grimes, Shelley; Jardine, Paul J et al. (2016) Single DNA molecule jamming and history-dependent dynamics during motor-driven viral packaging. Nat Phys 12:757-761
Yang, Teng-Chieh; Catalano, Carlos Enrique; Maluf, Nasib Karl (2015) Analytical Ultracentrifugation as a Tool to Study Nonspecific Protein-DNA Interactions. Methods Enzymol 562:305-30
Sippy, Jean; Patel, Priyal; Vahanian, Nicole et al. (2015) Genetics of critical contacts and clashes in the DNA packaging specificities of bacteriophages λ and 21. Virology 476:115-23
Feiss, Michael; Young Min, Jea; Sultana, Sawsan et al. (2015) DNA Packaging Specificity of Bacteriophage N15 with an Excursion into the Genetics of a Cohesive End Mismatch. PLoS One 10:e0141934
Feiss, Michael; Geyer, Henriette; Klingberg, Franco et al. (2015) Novel DNA packaging recognition in the unusual bacteriophage N15. Virology 482:260-8
Yang, Teng-Chieh; Ortiz, David; Nosaka, Lyn'Al et al. (2015) Thermodynamic Interrogation of the Assembly of a Viral Genome Packaging Motor Complex. Biophys J 109:1663-75
Rao, Venigalla B; Feiss, Michael (2015) Mechanisms of DNA Packaging by Large Double-Stranded DNA Viruses. Annu Rev Virol 2:351-78
Migliori, Amy D; Keller, Nicholas; Alam, Tanfis I et al. (2014) Evidence for an electrostatic mechanism of force generation by the bacteriophage T4 DNA packaging motor. Nat Commun 5:4173
Keller, Nicholas; delToro, Damian; Grimes, Shelley et al. (2014) Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage Phi29. Phys Rev Lett 112:248101

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