Probing the Universality and Function of Single dsDNA Viral Packaging Motors

描述(由申请人提供)：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操纵技术，我们有机会提取关于这些系统如何运行的前所未有的大量详细信息。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Lambda病毒是如何包装DNA的。其次，将研究马达获取化学能并将其转化为移动DNA的详细机制。最后，我们将探讨关键蛋白质对病毒存活的影响，以及对其包装DNA能力的影响。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.通过建立一种新的研究噬菌体A中DNA包装的光镊法，探索病毒DNA包装马达的普适性。 2.将通过测量ATP、ADP和PI依赖性、暂停分布和包装上的力测量来阐明Lambda病毒包装马达的机械力化学。 3.将研究对Lambda病毒包装至关重要的GPD和GPFI的具体作用。 这项研究将对公共卫生感兴趣，因为将研究的系统预计与相关哺乳动物病毒，如疱疹病毒、腺病毒和痘病毒有相似之处。它们被认为共享一个共同的包装机制，推测是由类似的分子马达驱动的。通过了解详细的机械力化学以及这些病毒生存所需的额外蛋白质，我们可能会设想新的策略来调节它们的传染性。

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.