Collaborative Research: Physical parameters controlling viral DNA packaging and ejection

合作研究：控制病毒 DNA 包装和排出的物理参数

• 批准号: 1716219
• 负责人: Douglas Smith
• 金额: $ 60万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1716219&HistoricalAwards=false
• 关键词: Collaborative; Research; Physical; parameters; controlling

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.

病毒是一种只在其他生物体的活细胞内复制的小型感染体，可以感染所有类型的生命形式。这项研究将增进对许多病毒生命周期中的关键步骤、病毒组装过程中DNA的包装和宿主细胞感染期间DNA的排出，以及这些过程如何依赖于紧密包装的DNA的物理行为的理解。该项目不仅将阐明病毒的基本生物学，还将阐明严密限制的DNA的物理学以及运输DNA的生物分子马达的调节。研究生和本科生将接受将物理技术应用于生物学研究的跨学科培训，并将开发新的实验实验室课程材料。将开展K-12推广活动，并将评估小学教材，包括科学书籍、工具包和游戏的质量和科学准确性，以向学校和博物馆提供在线建议。最近的研究表明，细菌病毒中双链DNA包装的动力学由紧密包装的DNA的动力学和能量学主导。在将要研究的生物状态中，DNA与自身的相互作用是排斥的，DNA只会非常缓慢地向平衡构象松弛。强大的力量可以抵抗DNA的限制，然后在感染宿主细胞的过程中帮助推动排出。噬菌体phi29将作为一个模型系统进行研究，并将使用光学镊子直接测量单个DNA分子的包装。包装动力学依赖于初始马达速度，依赖于ATP浓度，将被研究。较低的初始速度被认为可以减少非常不利的DNA构象的形成，从而减少动力学中的异质性，降低充填过程中的相对减速，并减少电机停顿、打滑和失速。一个需要检验的假说是，门静脉马达的变构调节通过降低马达速度来减少不利DNA构象的形成，从而帮助包装尽可能快地完成。包装动力学、作用力和DNA松弛如何依赖于温度和离子条件也将被研究。马达速度随着温度的升高而显著增加，而马达在充填过程中的减速取决于离子屏蔽条件。虽然较快的初始速度可能会在填充过程中导致更大的相对减慢，但温度升高或离子筛选可能会加速DNA松弛。包装条件和老化如何影响DNA喷射和病毒感染性也将被研究。在包装和老化过程中影响DNA构象的条件可能会影响DNA的排出。增加抵抗包装的力量或减少DNA松弛时间的条件可能会增强弹射。这些参数的生物学影响将通过检查它们对病毒感染性的影响来调查。

项目成果

Role of DNA-DNA sliding friction and nonequilibrium dynamics in viral genome ejection and packaging.

• DOI: 10.1093/nar/gkad582
• 发表时间: 2023-08-25
• 期刊: Nucleic acids research
• 影响因子: 14.9