Assembly of Picornaviral Replication Complexes

小核糖核酸病毒是一个小的正义单链RNA病毒家族，其引起广泛的疾病，每年的成本高达数亿美元。成员包括麻痹性脊髓灰质炎病毒和肠道病毒D 68，导致心脏病的科萨基B3病毒，以及导致一半以上普通感冒的鼻病毒。这些病毒共享一个生命周期，其中RNA复制和病毒组装发生在组装的大型膜锚定复制复合物中，并且复制过程由负责合成所有病毒RNA的病毒编码的RNA依赖性RNA聚合酶（3Dpol）驱动。该研究项目的重点是病毒复制中心的结构和组装以及病毒聚合酶的结构-功能研究，以了解控制延伸率和复制保真度的机制。我们将使用生物化学和结构生物学方法来研究3Dpol中使用的保真度检查点如何重新排列，因为这些聚合酶进化为支持大基因组冠状病毒复制，提供有关功能限制的新信息，以帮助我们了解病毒进化的途径。在第二个目标中，我们将阐明RNA与病毒蛋白酶相互作用并刺激病毒蛋白酶的机制，这一发现表明病毒RNA元件可以在病毒复制中心的背景下调节多蛋白加工。最后，我们将解决小核糖核酸病毒尿苷酰化复合物的结构，该复合物产生用于通过病毒3Dpol聚合酶进行所有RNA合成的VPg-pUpU引物。

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 paralyzing poliovirus and enterovirus D68, the heart disease causing coxsackie B3 virus, and rhinoviruses that cause more than half the occurrences of the common cold. These viruses share a life cycle where RNA replication and viral assembly occurs in large membrane anchored replication complexes assembled and 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 elongation rates and replication fidelity. We will use biochemical and structural biology approaches to study how the fidelity checkpoint used in 3Dpol was rearranged as these polymerases evolved to support large-genome coronavirus replication, providing novel information about functional constraints to help us understanding pathways for virus evolution. In a second aim we will elucidate the mechanisms whereby RNAs interact with and stimulate viral proteases, a finding that suggests viral RNA elements can regulated polyprotein processing the context of a viral replication center. Last, we will solve the structure of the picornaviral uridylylation complex that generates the VPg-pUpU primers used for all RNA synthesis by the viral 3Dpol polymerase.