Collaborative Research: Physical parameters controlling viral DNA packaging and ejection

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Viruses are small infectious agents that only replicate inside the living cells of other organisms and can infect all types of life forms. This research will improve the understanding of key steps in the life-cycle of many viruses, the packaging of the DNA during viral assembly and the ejection of the DNA during infection of a host cell, as well as how these processes depend on the physical behavior of the tightly packed DNA. The project will not only shed light on the fundamental biology of viruses, but also on the physics of tightly confined DNA and the regulation of biological molecular motors which transport DNA. Graduate and undergraduate students will receive interdisciplinary training in the application of physics techniques to biology research and new experimental laboratory course materials will also be developed. K-12 outreach activities will be conducted and grade-school educational materials, including science books, kits, and games, will be evaluated for quality and scientific accuracy to provide online recommendations to schools and museums.Recent studies have shown that the kinetics of the packaging of double stranded DNA in bacterial viruses is dominated by the dynamics and energetics of the tightly packed DNA. In the biological regime to be studied, with repulsive DNA-self interactions, the DNA relaxes only very slowly towards an equilibrium conformation. Large forces build that resist DNA confinement and later help drive ejection during infection of a host cell. Bacteriophage phi29 will be studied as a model system and the packaging of single DNA molecules will be directly measured with optical tweezers. The dependence of packaging kinetics on initial motor velocity, dependent on ATP concentration, will be studied. Lower initial velocity is hypothesized to reduce formation of highly unfavorable DNA conformations, yielding decreased heterogeneity in the dynamics, lower relative slowing during filling, and less motor pausing, slipping, and stalling. A hypothesis to be tested is that allosteric regulation of the portal motor helps packaging complete as fast as possible by throttling down the motor velocity to mitigate formation of unfavorable DNA conformations. How packaging kinetics, force, and DNA relaxation depend on temperature and ionic conditions will also be studied. Motor velocity increases significantly with temperature, and motor slowing during filling depends on ionic screening conditions. Whereas faster initial velocity may cause greater relative slowing during filling, increased temperature or ionic screening may accelerate DNA relaxation. How packaging conditions and aging affect DNA ejection and viral infectivity will also be studied. Conditions affecting the DNA conformation during packaging, and aging, may impact DNA ejection. Conditions that increase forces resisting packaging or decrease DNA relaxation time may enhance ejection. The biological impact of these parameters will be investigated by examining their effects on virus infectivity.