The Molecular Structure of the RNA Polymerase Portal of a Bacteriophage-Reovirus

描述(申请人提供)：我们的研究旨在了解构成囊状病毒RNA聚合酶门户的蛋白质之间存在的相互作用。这台分子机器选择、打包和复制病毒RNA基因组片段。一个入口位于12面聚合酶复合体(PX)的每个顶点上，PX是形成病毒核心的结构。该噬菌体家族构成了一种独特的细菌呼肠孤病毒，为它们在临床上有重要意义的近亲呼肠孤病毒科提供了一个简单而重要的分子模型。我们的总体目标是利用冷冻电子显微镜(Cryo-EM)为呼肠孤病毒门脉装置建立一个详细的结构模型。然后，我们将更好地了解它们是如何复制和打包双链RNA(DsRNA)基因组的。这一知识对于设计针对致病性呼肠孤病毒，特别是轮状病毒的抗病毒药物至关重要，轮状病毒是第三世界国家儿童发病率的重要原因。我的发展和职业目标是聘请一支合格的研究团队，在纽约城市学院(CCNY)医学院研究具有临床意义的病毒的分子生物学和结构。囊状病毒模型提供了一个出色的入门病毒项目，该项目利用了CCNY校区最近建立的分子和结构生物学资源，特别是纽约州科学、技术和创新基金会(NYSTAR)资助的纽约结构生物学中心(NYSBC)。Gottlieb实验室的工作人员利用了这个新设施的硬件和软件，并发表了该中心的第一篇电子冷冻断层扫描论文。我们正在完成第二份电子冷冻显微镜论文，这份论文构成了这一提议的初步研究。我们最近建立了一种新的光学方法来检测病毒蛋白在感染过程中发生的动态构象形状变化。这项研究是与CCNY园区超快光谱与激光研究所(IUSL)的物理学家合作进行的。我们在研究计划中包括了一个目标，旨在完善我们用于分析具有生物化学活性的囊状病毒PX的光学分析方法。众所周知，Px在病毒RNA包装和复制过程中会从根本上扩张。我们相信，我们研究提案的这一部分很好地符合当前项目的目标。我们预计，我们发现的病毒蛋白的光学性质适用于人类病原体，特别是流感。 公共卫生相关性：疾病控制中心(CDC)指出，A组轮状病毒是导致青少年腹泻的主要原因，导致美国每年数千人住院，全球近100万人死亡。目前还没有治疗这种疾病的抗病毒配方，目前的疫苗也不是100%有效。我们的研究利用了囊状病毒，这是轮状病毒的一种，只在细菌细胞中复制。这是一个我们可以非常详细地研究的系统，以了解病毒复制的机制，并为开发抗轮状病毒化合物提供关键信息。

DESCRIPTION (provided by applicant): Our study is directed towards understanding the interactions that exist among the proteins that constitute the RNA polymerase portal of the cystoviruses. This molecular machine selects, packages, and replicates the viral RNA genome segments. One portal is located on each vertex of the 12-sided polymerase complex (PX) a structure that forms the viral core. This bacteriophage family constitutes a unique bacterial reovirus that serves as a simple and important molecular model for their clinically significant cousins, the Reoviridae. Our overall objective is to establish a detailed structural model for the reoviruses portal apparatus using cryo-electron microscopy (cryo-EM). We will then better understand how they replicate and package their double-stranded RNA (dsRNA) genomes. This knowledge is of critical importance in the design of antiviral pharmaceuticals for pathogenic reoviruses, in particular rotavirus, a significant cause of childhood morbidity in third world countries. My developmental and career objective is to employ a qualified research team that studies the molecular biology and structure of clinically significant viruses at the City College of New York (CCNY) Medical School. The cystovirus model offers an excellent introductory virus project that utilizes the resources for molecular and structural biology recently established at the CCNY campus in particular the New York State Foundation for Science, Technology and Innovation (NYSTAR) funded New York Structural Biology Center (NYSBC). The Gottlieb laboratory personnel have utilized the hardware and software at this new facility and have published the center's first electron-cryo- tomography paper. We are completing a second electron cryo-microscopy paper that constitutes the preliminary study for this proposal. We have recently established a new optical method to examine the dynamic conformational shape changes that occur in viral proteins during the infection process. This research is performed in collaboration with physicists at the Institute for Ultrafast Spectroscopy and Lasers (IUSL) on the CCNY campus. We have included an aim in the research plan directed towards perfecting our optical assays for the analysis of biochemically active cystovirus PX. The PX is known to radically expand during viral RNA packaging and replication. We believe that this portion of our research proposal fits well within the goals of the current project. We anticipate that the optical qualities of viral proteins that we discover are applicable to human pathogens, in particular influenza. PUBLIC HEALTH RELEVANCE: Group A rotaviruses, as noted by the Centers for Disease Control (CDC), are the major cause of juvenile diarrhea leading to several thousand hospitalizations annually in the United States and close to a million deaths worldwide. There is no antiviral formulation to treat this illness and the current vaccine is not 100% effective. Our research utilizes cystoviruses, a type of rotavirus, which only replicate in bacteria cells. This is a system that we can study in great detail to understand the mechanisms of virus reproduction, and provide information critical to the development of anti-rotavirus compounds.