Assembly of Picornaviral Replication Complexes

描述(申请人提供)：小核糖核酸病毒是一类小的正向单链RNA病毒，每年造成数亿美元的疾病。成员包括急性甲型肝炎病毒、导致心脏病的柯萨奇B3病毒、导致一半以上普通感冒发生的鼻病毒，以及瘫痪的脊髓灰质炎病毒。这些病毒具有共同的生命周期，它们的RNA复制和病毒组装发生在大的膜锚定复制复合体中，这些复制复合体组装在来自内质网的小泡的表面。复制过程是由病毒编码的依赖RNA的RNA聚合酶3Dpol蛋白驱动的，该蛋白负责所有病毒RNA的合成。像所有的小核糖核酸病毒蛋白一样，聚合酶是由单个大的病毒多蛋白的蛋白水解性裂解而产生的。在几种小核糖核酸病毒中有越来越多的证据表明，聚合酶及其直接前体直接负责这些复制中心的组装。脊髓灰质炎病毒的3Dpol聚合酶是研究最深入的微小核糖核酸病毒，已被证明沿着蛋白质-蛋白质界面组装成大的片状结构，最初在3Dpol的部分晶体结构中发现了这种结构。我们已经在2.0A分辨率下解析了3Dpol的完整晶体结构，并发现该酶需要一个自由的N末端来正确折叠活性部位，为加工依赖的聚合酶的激活提供了分子基础。我们正在继续我们的脊髓灰质炎病毒蛋白质的结构研究，通过进一步表征3Dpol的构象灵活性，并扩展到确定蛋白质-蛋白质界面在3CDpro和其他前体蛋白质组装中的作用。这些结果将对这组重要病原体的复制产生基本的见解。

DESCRIPTION (provided by applicant): 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 common life cycle where their 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, the 3Dpol protein, that is responsible the synthesis of all viral RNA. Like all picornaviral proteins, the polymerase is generated by proteolytic cleavage of a single large viral polyprotein. There is mounting evidence in several picornaviruses that the polymerase and its immediate precursors are directly responsible for the assembly of these replication centers. The 3Dpol polymerase of poliovirus, the best studied of the picornaviruses, has been shown to assemble into large sheet structures along a protein-protein interface that was initially identified in a partial crystal structure of 3Dpol. We have solved the complete crystal structure of 3Dpol at 2.0 A resolution and discovered that the enzyme requires a free N-terminus to properly fold the active site, providing a molecular basis for the processing dependent activation of the polymerase. We are continuing our structural studies of the poliovirus proteins by further characterizing the conformational flexibility of 3Dpol and expanding into determining the role of protein-protein interfaces in the assembly of 3CDpro and other precursor proteins. The results will yield fundamental insights into the replication of this important group of pathogens.