Assembly of Picornaviral Replication Complexes

小核糖核酸病毒是一类小的正向单链rna病毒家族，可引起广泛的 每年的费用高达数亿美元。成员包括急性甲型肝炎病毒， 引起柯萨奇B3病毒的心脏病，鼻病毒导致一半以上的 普通感冒和麻痹的脊髓灰质炎病毒。这些病毒共享一个生命周期，即RNA复制和病毒 组装发生在组装在囊泡表面的大的膜锚定的复制复合体中 来源于内质网。复制过程是由依赖病毒编码的RNA驱动的 负责合成所有病毒RNA的RNA聚合酶(3Dpol)。本课题研究的重点是 病毒复制中心的结构和组装及其结构-功能研究 聚合酶以了解控制延伸率和复制保真度的机制。我们有 解决了3Dpol的多种晶体结构作为与结合的RNA的伸长络合物和这些结构 揭示了RdRPs关闭其催化活性部位以响应核苷酸的新机制 有约束力的。在即将到来的项目期间，我们将使用生物物理和结构生物学方法来进一步 3DPoll对RNA易位的机理理解和解决病毒起始和释放的结构 尿苷基化复合体。我们还将使用单分子显微镜和粒子跟踪方法直接 可视化病毒复制复合体在支持的脂质双层上的体外组装。

The picornaviruses are a family of small positive sense single stranded RNA viruses that cause a wide range of diseases at an annual cost well into the hundreds of million dollars. Members include acute hepatitis A virus, the heart disease causing coxsackie B3 virus, rhinoviruses that cause more than half the occurrences of the common cold, and the paralyzing poliovirus. These viruses share a life cycle where RNA replication and viral assembly occurs in large membrane anchored replication complexes assembled on the surfaces of vesicles derived from the endoplasmic reticulum. The replication process is driven by a virally encoded RNA dependent RNA polymerase (3Dpol) that is responsible for the synthesis of all viral RNA. This research project is focused on the structure and assembly of the viral replication centers and on structure-function studies of the viral polymerases to understand mechanisms that control of elongation rates and replication fidelity. We have solved multiple crystal structures of 3Dpol as elongation complexes with bound RNA and these structures revealed a novel mechanism by which RdRPs close their active sites for catalysis in response to nucleotide binding. In this upcoming project period we will use biophysical and structural biology methods to further our mechanistic understanding of RNA translocation by 3Dpol and to solve the structures of viral initiation and uridylylation complexes. We will also use single molecule microscopy and particle tracking methods to directly visualize the in vitro assembly of viral replication complexes onto supported lipid bilayers.