Poliovirus 3D--Cooperative RNA Binding & Polymerization

脊髓灰质炎病毒 3D——RNA 协同结合

• 批准号: 6706235
• 负责人: Karla Kirkegaard
• 金额: $ 32万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6706235
• 关键词: DNA directed RNA polymerase; SDS polyacrylamide gel electrophoresis; chemical binding; electron microscopy; freeze etching; poliovirus; polymerase chain reaction; polymerization; replicase; virus RNA; virus replication

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.

描述（由申请方提供）：正链RNA病毒（如脊髓灰质炎病毒）在其真核宿主的细胞内膜上复制其基因组。脊髓灰质炎病毒的RNA依赖性RNA聚合酶3D可以通过与膜相关病毒蛋白3AB相互作用连接到细胞内膜。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确定。 从感染细胞中分离的膜囊泡含有与其表面上存在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RNA复制发生在聚合酶催化晶格上的假设仍有待明确验证。在特定目标1中，将使用一组定义的温度敏感性病毒来监测感染期间3AB和3D聚合酶向膜的募集，以鉴定该募集所需的分子间接触，通过冷冻蚀刻电子显微镜观察所得复合物，并检测聚合酶寡聚化的遗传后果。在特定目标2中，描述了鉴定结合模板RNA沿着聚合酶寡聚体的路径的测定和探测长RNA模板可被固定化聚合酶寡聚体复制的机制的测试。在特定目标3中，探索了蛋白质引物3B结合到聚合酶表面的令人惊讶的发现，该聚合酶表面不同于用其他聚合酶观察到的核酸引物的已知结合位点。实验，以确定如何聚合酶可以催化磷酸二酯键的形成结合在两个不同的网站上的基板，以及这是否需要形成含聚合酶的低聚物。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期间使用寡聚晶格。最近也有报道，人类端粒酶和丙型肝炎病毒RNA依赖的RNA聚合酶经历寡聚化，这可能是其功能所必需的。因此，这些研究与脊髓灰质炎病毒，其中丰富的结构，细胞生物学和遗传工具的存在，可以提供一个范例，膜相关的核酸合成在许多系统中。最后，我们提供了选择寡聚蛋白作为抗病毒靶点的遗传学依据。