Poliovirus 3D--Cooperative RNA Binding & Polymerization

描述(申请人提供)：正链RNA病毒，如脊髓灰质炎病毒，在其真核宿主的细胞膜上复制其基因组。脊髓灰质炎病毒依赖于RNA的RNA聚合酶3D可以通过与膜相关的病毒蛋白3AB相互作用连接到细胞膜上。通过X射线结晶学、定点突变和电子显微镜确定了聚合酶3D与3AB结合的表面，以及聚合酶齐聚形成大的平面晶格的表面。从感染细胞分离的膜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复制是在聚合酶的催化晶格上发生的这一假设仍有待明确测试。在具体目标1中，将使用一组明确的温度敏感病毒来监测感染期间3AB和3D聚合酶在膜上的募集情况，确定这一募集所需的分子间接触，通过冷冻蚀刻电子显微镜观察得到的复合体，并测试聚合酶寡聚的遗传后果。在具体目标2中，描述了识别结合模板RNA沿聚合酶低聚物路径的方法，以及探索固定聚合酶低聚物复制长RNA模板的机制的测试。在具体目标3中，探索了令人惊讶的发现，即蛋白质引物3B与聚合酶表面结合，不同于与其他聚合酶观察到的核酸引物的已知结合部位。实验是为了确定聚合酶如何催化结合在两个不同位置的底物的磷酸二酯键的形成，以及这是否需要形成含有聚合酶的寡聚体。对于另一个基因组，即DNA噬菌体phi29，有人建议在膜相关复制过程中使用低聚格子。最近也有报道，人类端粒酶和丙型肝炎病毒RNA依赖的RNA聚合酶经历了可能需要的寡聚，这可能是它们发挥功能所必需的。因此，这些关于脊髓灰质炎病毒的研究，有丰富的结构、细胞生物学和遗传工具，可能为许多系统中的膜相关核酸合成提供一个范例。最后，我们提供了选择寡聚蛋白作为抗病毒靶点的遗传学基础。

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.