Probing the Universality and Function of Single dsDNA Viral Packaging Motors

描述（由申请方提供）：本提案的目标是帮助更好地了解病毒包装马达的功能，并找出对其功能至关重要的组件的作用。使用单一病毒和DNA操作技术，我们有机会提取前所未有的大量关于这些系统如何运作的详细信息。此外，通过探索不同的病毒包装系统，我们可以测试某些类别的病毒是否都使用相同的机制工作。首先，将开发一种实验技术来研究λ病毒如何包装DNA。其次，将研究马达可以吸收化学能并将其转化为移动DNA的详细机制。最后，将探讨关键蛋白质对该病毒生存的影响，以及其包装DNA的能力。从这些实验中收集的基本信息可能有助于抗病毒药物的合理设计。 具体目标： 1.本研究将通过开发一种新的光镊技术来研究噬菌体A中DNA的包装，探索病毒DNA包装马达的普遍性。运动力，DNA易位测量，以及它们对生化条件的依赖性将扩展到A。 2.的λ病毒包装电机的机械化学将阐明通过测量ATP，ADP，和Pi的依赖性，暂停分布，和力的测量包装。 3.将研究对于λ病毒包装至关重要的蛋白质gpD和gpFI的具体作用。 这项研究将对公共卫生感兴趣，因为预计将研究的系统与相关的哺乳动物病毒（如疱疹病毒，腺病毒和痘病毒）具有相似性。它们被认为有一个共同的包装机制，大概是由一个类似的分子发动机驱动的。通过了解详细的机械化学以及这些病毒生存所必需的其他蛋白质，我们可以设想新的策略来调节它们的感染性。

DESCRIPTION (provided by applicant): The goals of this proposal are to help bring a better understanding of how viral packaging motors function and to find out the role of the components critical to their function. Using single virus and DNA manipulation techniques, we have the opportunity to extract an unprecedented amount of detailed information on how these systems operate. Furthermore, by exploring different viral packaging systems we can test whether certain classes of viruses all work using the same mechanisms. First, an experimental technique will be developed to investigate how lambda viruses package DNA. Second, the detailed mechanism in which the motor can take chemical energy and convert it to moving DNA will be investigated. Finally, the effect of critical proteins to the survival of this virus on its ability to package DNA will be explored. The fundamental information gathered from these experiments may aid the rational design of antiviral drugs. Specific Aims: 1. The universality of viral DNA packaging motors will be explored by developing a new optical tweezers assay for studying DNA packaging in bacteriophage A. Motor force, DNA translocation measurements, and their dependence on biochemical conditions will be extended to A. 2. The mechanochemistry of the lambda viral packaging motor will be elucidated by measuring ATP, ADP, and Pi dependence, pause distributions, and force measurements on packaging. 3. The specific roles of gpD and gpFI, proteins critical for lambda viral packaging, will be investigated. This research will be of interest to public health because the system that will be studied is expected to have similarities to relevant mammalian viruses such as herpesviruses, adenoviruses, and poxviruses. They are thought to share a common packaging mechanism, presumably powered by a similar molecular motor. By understanding the detailed mechanochemistry as well as the additional proteins necessary for the survival of these viruses, we may envision new strategies to regulate their infectivity.