Dynamics and Energetics of Viral DNA Packaging and Ejection

该奖项是根据2009年美国复苏和再投资法案(公法111-5)资助的。在这个项目中，PI将使用phi29作为模型系统来研究病毒基因组包装的物理原理。这项研究将使用以前开发的基于光学镊子的技术来实时可视化病毒包装过程。该项目将研究以下对病毒DNA包装和喷射的影响：(1)多胺离子对DNA的筛选：寄主细胞多胺，包括腐胺2+、精胺3+和精胺4+，预计将通过影响DNA力学和静电学、填充力和运动功能来显著影响DNA包装动力学。(2)非平衡动力学：DNA填充力将通过研究不同浓度的ATP引起的运动速度变化引起的内力的时间依赖性来研究，以及在不同的衣壳填充水平下由不可水解的ATP类似物引起的包装暂停。包装动力学和力的潜在异质性，正如最近的理论模拟所预测的那样，也将用单分子测量的系综进行研究。(3)衣壳形状：不同的病毒衣壳形状不同，可能会影响DNA包装的动态。(4)DNA结合蛋白：PI将研究与DNA相互作用的蛋白质“路障”的影响，并在包装马达上产生额外的负载。课程内容包括聚合物(DNA)的构象和动力学、分子马达功能、离子筛选、蛋白质-DNA相互作用和病毒组装原理。研究生和本科生将由不同背景的PI指导，学生将进行涉及物理和生物科学的概念和方法的研究。本科生将参与这项研究，并将开发新的本科物理和生物课程材料。还将与圣地亚哥的R.H.舰队科学博物馆合作开发K-12科学教育材料。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).In this project the PI will study the physical principles of viral genome packaging using phi29 as a model system. The study will use previously developed technique based on optical tweezers for real time visualization of viral packaging processes. The project will examine the following effects on viral DNA packaging and ejection: (1) Screening of DNA by polyamine ions: Host cell polyamines including putrescine2+, spermidine3+, and spermine4+ are expected to substantially influence DNA packaging dynamics by affecting DNA mechanics and electrostatics, packing forces, and motor function. (2) Non-equilibrium dynamics: DNA packing forces will be investigated by studying the time-dependence of the internal forces resulting from changes in motor velocity induced by varying ATP concentration, and pauses in packaging induced with non-hydrolyzable ATP analogs at various capsid filling levels. Potential heterogeneity in packaging dynamics and force, as predicted in recent theoretical simulations, will also be investigated with ensembles of single molecule measurements. (3) Capsid shape: Different viruses have different capsid shapes that may affect DNA packaging dynamics. (4) DNA binding proteins: The PI will study the effect of protein "roadblocks" that interact with the DNA and create an additional load on the packaging motor. Topics of broad interest in physics and biology will be addressed, including polymer (DNA) conformation and dynamics, molecular motor function, ionic screening, protein-DNA interactions, and principles of viral assembly. Graduate and undergraduate students will be supervised by PIs with varying backgrounds and the students will perform research involving concepts and methods from the physical and biological sciences. Undergraduates will participate in this research and new undergraduate physics and biology course materials will be developed. Materials for K-12 science education will also be developed in collaboration with the R. H. Fleet Science Museum in San Diego.