Poliovirus 3D--Cooperative RNA Binding & Polymerization

描述（由申请人提供）：正链RNA病毒（如脊髓灰质炎病毒）在真核宿主的细胞膜上复制其基因组。脊髓灰质炎病毒依赖RNA的RNA聚合酶3D可通过与膜相关病毒蛋白3AB的相互作用与细胞膜连接。聚合酶3D与3AB结合的表面，以及聚合酶寡聚形成大平面晶格的表面，已经通过x射线晶体学、定点诱变和电子显微镜来定义。从感染细胞中分离出的膜状囊泡含有与其表面存在的二维聚合酶阵列一致的结构。然而，脊髓灰质炎病毒感染细胞中的RNA复制发生在聚合酶的催化晶格上的假设仍有待明确验证。在Specific Aim 1中，一组定义的温度敏感型病毒将用于监测感染期间3AB和3D聚合酶在膜上的招募，确定这种招募所需的分子间接触，通过冷冻刻蚀电子显微镜观察所产生的复合物，并测试聚合酶寡聚化的遗传后果。在特异性目标2中，描述了确定结合模板RNA沿着聚合酶低聚物的路径和检测长RNA模板可以通过固定化聚合酶低聚物复制的机制的方法。在Specific Aim 3中，我们探索了一个令人惊讶的发现，即蛋白质引物3B与已知的核酸引物与其他聚合酶的结合位点不同，它与聚合酶表面结合。实验提出，以确定如何聚合酶可以催化磷酸二酯键形成的底物结合在两个不同的位点，这是否需要形成含有聚合酶的低聚物。对于另一个基因组，DNA噬菌体phi29的基因组，已经提出在膜相关复制过程中使用寡聚晶格。最近也有报道称，人类端粒酶和丙型肝炎病毒RNA依赖的RNA聚合酶经历寡聚化，这可能是其功能所必需的。因此，存在丰富的结构、细胞生物学和遗传学工具的脊髓灰质炎病毒的这些研究可能为许多系统中的膜相关核酸合成提供范例。最后，我们提供了选择寡聚蛋白作为抗病毒靶点的遗传原理。

DESCRIPTION (provided by applicant): Positive-strand RNA viruses such as poliovirus replicate their genomes on intracellular membranes of their eukaryotic hosts. The RNA-dependent RNA polymerase of poliovirus, 3D, can be linked to intracellular membranes via interaction with a membrane-associated viral protein, 3AB. The surfaces by which polymerase 3D binds to 3AB, and through which the polymerase oligomerizes to form large planar lattices, have been defined by x-ray crystallography, site-directed mutagenesis and electron microscopy. Membranous vesicles isolated from infected cells contain structures consistent with the presence of two-dimensional polymerase arrays on their surfaces. However, the hypothesis that RNA replication in poliovirus-infected cells occurs on catalytic lattices of polymerase remains to be tested explicitly. In Specific Aim 1, a collection of defined temperature-sensitive viruses will be used to monitor the recruitment of 3AB and 3D polymerase to membranes during infection, to identify the intermolecular contacts required for this recruitment, to visualize the resulting complexes by freeze-etch electron microscopy, and to test the genetic consequences of polymerase oligomerization. In Specific Aim 2, assays to identify the path of bound template RNA along a polymerase oligomer and tests to probe the mechanism by which long RNA templates can be copied by immobilized polymerase oligomers are described. In Specific Aim 3, the surprising finding that the protein primer, 3B, binds to a polymerase surface distinct from the known binding sites of nucleic acid primers observed with other polymerases, is explored. Experiments are presented to determine how a polymerase can catalyze phosphodiester bond formation for substrates bound at two different sites, and whether this requires the formation of polymerase-containing oligomers. For one other genome, that of DNA bacteriophage phi29, the use of oligomeric lattices during membrane-associated replication has been proposed. It has also been reported recently that human telomerase and hepatitis C virus RNA-dependent RNA polymerase undergo oligomerization that is likely to be required for their function. Thus, these studies with poliovirus, for which an abundance of structural, cell biological and genetic tools exists, may provide a paradigm for membrane-associated nucleic acid synthesis in many systems. Finally, we provide a genetic rationale for choosing oligomeric proteins as antiviral targets.