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.

病毒是小型传染原，仅在其他生物体的活细胞内复制，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关键步骤、病毒组装过程中 DNA 包装和宿主细胞感染过程中 DNA 喷射的理解，以及这些过程如何依赖于紧密包装的 DNA 的物理行为。该项目不仅将揭示病毒的基本生物学，还将揭示严格限制的 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物理技术应用于生物学研究的跨学科培训，并且还将开发新的实验实验室课程材料。将开展 K-12 外展活动，并对小学教育材料（包括科学书籍、套件和游戏）进行质量和科学准确性评估，以便为学校和博物馆提供在线推荐。最近的研究表明，细菌病毒中双链 DNA 的包装动力学由紧密包装的 DNA 的动力学和能量主导。在要研究的生物体系中，由于 DNA 与自身的排斥相互作用，DNA 只能非常缓慢地松弛到平衡构象。产生的巨大力量可以抵抗 DNA 限制，并在宿主细胞感染期间帮助驱动喷射。噬菌体 phi29 将作为模型系统进行研究，并且将用光镊直接测量单个 DNA 分子的包装。将研究包装动力学对初始电机速度（取决于 ATP 浓度）的依赖性。假设较低的初始速度可以减少高度不利的 DNA 构象的形成，从而降低动力学的异质性，降低填充过程中的相对减慢，并减少运动暂停、打滑和失速。需要测试的一个假设是，门电机的变构调节通过降低电机速度来减轻不利 DNA 构象的形成，从而帮助包装尽可能快地完成。包装动力学、力和 DNA 松弛如何依赖于温度和离子条件也将被研究。电机速度随着温度的升高而显着增加，填充过程中电机的减速取决于离子筛选条件。虽然较快的初始速度可能会导致填充期间相对较慢，但升高温度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。包装和老化过程中影响 DNA 构象的条件可能会影响 DNA 喷射。增加抵抗包装力或减少 DNA 松弛时间的条件可能会增强弹出。将通过检查这些参数对病毒感染性的影响来研究这些参数的生物学影响。

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