Assembly of Picornaviral Replication Complexes

描述（由申请人提供）：小核糖核酸病毒是一种小的阳性单链RNA病毒家族，每年造成数亿美元的费用，导致各种疾病。这些病毒包括急性甲型肝炎病毒、引起心脏疾病的柯萨奇B3病毒、导致半数以上普通感冒的鼻病毒，以及麻痹性脊髓灰质炎病毒。这些病毒有一个共同的生命周期，它们的RNA复制和病毒组装发生在内质网囊泡表面组装的大型膜锚定复制复合体中。复制过程是由病毒编码的依赖RNA的RNA聚合酶，即3Dpol蛋白驱动的，它负责所有病毒RNA的合成。像所有小核糖核酸病毒蛋白一样，聚合酶是由单个大病毒多蛋白的蛋白水解裂解产生的。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聚合酶是研究得最好的小核糖核酸病毒，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的部分晶体结构中被鉴定出来。我们在2.0 A分辨率下求解了3Dpol的完整晶体结构，发现该酶需要一个游离的n端才能正确折叠活性位点，为该聚合酶的加工依赖性激活提供了分子基础。我们正在继续对脊髓灰质炎病毒蛋白的结构研究，进一步表征3Dpol的构象灵活性，并扩展到确定蛋白-蛋白界面在3CDpro和其他前体蛋白组装中的作用。这些结果将对这一重要病原体群的复制产生根本性的见解。

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.