Mechanisms of Viral DNA Packaging

 描述(申请人提供)：在自组装过程中，许多噬菌体和一些真核病毒--包括人类疱疹病毒和腺病毒--在强大的ATP依赖的马达的作用下将它们的双链DNA基因组包装成预先形成的衣壳。由于人们认为这些病毒使用类似的机制来包装DNA，因此基因组包装过程是广谱抗病毒药物开发的一个有前途的目标。噬菌体29的包装马达是研究病毒包装的理想模型系统，因为它具有强大的体外包装试验和广泛的遗传、生化、结构和单分子特性。由于这个马达由属于环NTPase超家族的五聚体ATPase环组成，它的研究也将有助于揭示该家族中负责大量细胞功能的其他成员的运作，如ATP合成、染色体分离、双链解离和蛋白质解离。我们之前的单分子研究使我们能够为29包装电机建立一个全面的机械力化学模型，并为我们提供了一个独特的机会，以前所未有的细节解决与电机操作有关的基本机械问题。在这项应用中，我们将重点放在这台电机高度协调和精细调节的物理基础上。具体地说，我们建议：(1)通过监测应激条件下的野生型马达和缺乏协调表型的突变马达来剖析亚基间协调的机制；(2)表征DNA与马达之间不同类型接触的性质和强度，以及这些接触在马达操作中的作用；(3)绘制充满DNA的衣壳和包装ATPase之间的通讯路径，并将马达复合体的构象动力学与其包装行为关联起来。为了进行这些研究，我们将利用实验室中最先进的单分子仪器，包括高分辨率的双陷阱光学镊子和下一代荧光力混合显微镜。单分子生物物理测量的结果将通过已建立的合作关系与遗传、生化和结构研究相证实。这些跨学科的努力将使我们更接近于全面了解病毒包装过程，并为病毒感染的治疗干预提供新的机会。

 DESCRIPTION (provided by applicant): During their self-assembly many bacteriophages and a number of eukaryotic viruses - including human herpesviruses and adenoviruses - package their double-stranded DNA genomes into pre-formed capsids by the action of a powerful ATP-dependent motor. Since it is believed that these viruses employ similar mechanisms to package DNA, the genome packaging process is a promising target for broad-spectrum anti- viral drug development. The packaging motor of bacteriophage 29 is an ideal model system to investigate viral packaging due to a robust in-vitro packaging assay and extensive genetic, biochemical, structural, and single-molecule characterizations. Since this motor is comprised of a pentameric ring of ATPases that belong to the ASCE superfamily of ring NTPases, its study will also shed light on the operation of other members of this family that are responsible for a large number of cellular functions, such as ATP synthesis, chromosomal segregation, duplex unwinding, and protein unfolding. Our previous single-molecule studies allow us to build a comprehensive mechanochemical model for the  29 packaging motor and provide us with a unique opportunity to tackle fundamental mechanistic questions regarding motor operation with unprecendeted detail. In this application, we focus on the physical basis for the high degree of coordination and exquisite regulation observed in this motor. Specifically, we propose to: (1) dissect the mechanism of intersubunit coordination by monitoring wild-type motors under stressed conditions and mutant motors with deficient coordination phenotypes; (2) characterize the nature and strength of different types of contacts made between the DNA and the motor and the roles of these contacts in motor operation; (3) map the communication pathway between the DNA-filled capsid and the packaging ATPase and correlate the conformational dynamics of the motor complex to its packaging behavior. To carry out these studies, we will take advantage of state-of-the-art single-molecule instrumentation housed in our laboratory, including high-resolution dual-trap optical tweezers and a next- generation fluorescence-force hybrid microscope. Results of single-molecule biophysical measurements will be corroborated with genetic, biochemical, and structural studies through established collaborations. These interdisciplinary efforts will bring us closer toward a complete understanding of the viral packaging process and provide new opportunities for therapeutic intervention of viral infection.