Mechanisms of Viral DNA Packaging

 描述（由申请人提供）：在其自组装过程中，许多噬菌体和许多真核病毒（包括人疱疹病毒和腺病毒）通过强大的ATP依赖性马达的作用将其双链DNA基因组包装成预先形成的衣壳。由于认为这些病毒采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有希望的靶标。噬菌体B129的包装马达是研究病毒包装的理想模型系统，这是由于稳健的体外包装测定和广泛的遗传、生化、结构和单分子表征。由于该马达由属于ASCE环NTPases超家族的ATP酶的五聚环组成，因此其研究也将揭示该家族的其他成员的操作，这些成员负责大量的细胞功能，如ATP合成，染色体分离，双链体解旋和蛋白质解折叠。我们以前的单分子研究使我们能够为2019包装电机建立一个全面的机械化学模型，并为我们提供了一个独特的机会来解决有关电机操作的基本机械问题。在这个应用程序中，我们专注于在这个电机中观察到的高度协调和精致调节的物理基础。具体而言，我们建议：（1）通过监测应激条件下的野生型马达和具有缺陷协调表型的突变马达来剖析亚基间协调的机制;（2）表征DNA和马达之间形成的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DNA填充的衣壳和包装ATP酶之间的通讯路径，并将马达复合物的构象动力学与其包装行为相关联。为了进行这些研究，我们将利用我们实验室中最先进的单分子仪器，包括高分辨率双阱光镊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与遗传、生物化学和结构研究相证实。这些跨学科的努力将使我们更接近于对病毒包装过程的完整理解，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.