INTERACTIONS WITHIN RABIES VIRUS RNP COMPLEX

狂犬病病毒 RNP 复合物内的相互作用

• 批准号: 2067995
• 负责人: ZHEN F FU
• 金额: $ 10.66万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2067995
• 关键词: RNase protection assay; SDS polyacrylamide gel electrophoresis; X ray crystallography; chemical binding; crystallization; gene expression; genome; immunoprecipitation; intermolecular interaction; mutant; phosphoproteins; polymerase chain reaction; protein purification; protein sequence; protein signal sequence; protein structure; rabies virus; ribonucleoproteins; transfection /expression vector; virus RNA; virus genetics

Rabies virus ribonucleoprotein (RNP) complex which is comprised of the genomic negative strand RNA surrounded by nucleoprotein (N), phosphoprotein (NS), and RNA polymerase (L), is capable of initiating infection in cells, and is therefore the necessary unit for both transcription and replication. The N and NS proteins are the major components of the RNP complex, and are thought to play important roles in the process of transcription and replication of viral RNA by interacting with each other, as well as with L protein and viral RNA. As a first step toward a better understanding of rabies virus transcription and replication, I propose to study the structure of rabies virus N protein and its interaction with NS protein and genomic RNA, and to locate the regions on N protein that interact with these components. The N and NS proteins of rabies virus ERA strain will be expressed in insect cells, using baculovirus vectors, and purified individually or as complexes. The detailed structure of these proteins or protein complexes will be studied by crystallization and X-ray diffraction. The interaction of N and NS proteins will be studied by mixing the purified N and NS proteins at different ratios and analyzing the complexes formed. The interaction of N protein with viral RNA will be investigated by assessing the ability of expressed N protein to encapsidate in vitro synthesized rabies virus leader RNA, and thereby protect it from ribonuclease digestion. The interaction between rabies virus N and NS proteins and between N protein and genomic RNA will also be studied by simultaneously expressing the components in the newly available one step in vitro transcription/translation system. The regions of these components (N protein, NS protein, genomic RNA) that are involved in the interaction will also be identified by expressing protein mutants with C-terminal truncations and internal deletions, and studying the binding activity of these mutagenized proteins.

狂犬病病毒核糖核蛋白(RNP)复合体，由 被核蛋白(N)包围的基因组负链RNA， 磷酸蛋白(NS)和核糖核酸聚合酶(L)能够启动 在细胞中的感染，因此是两者的必要单位 转录和复制。N蛋白和NS蛋白是主要的 是RNP复合体的组成部分，被认为在 病毒核糖核酸相互作用的转录和复制过程 与L蛋白和病毒核糖核酸的相互作用。作为第一次 朝着更好地理解狂犬病病毒转录和 复制，我建议研究狂犬病病毒N蛋白的结构 以及它与NS蛋白和基因组RNA的相互作用，并定位 N蛋白上与这些成分相互作用的区域。N和NS 狂犬病病毒ERA株的蛋白将在昆虫细胞中表达， 使用杆状病毒载体，并单独或作为复合体进行纯化。 这些蛋白质或蛋白质复合体的详细结构将是 用结晶和X-射线衍射法进行了研究。N的相互作用 将纯化的N蛋白和NS蛋白混合，对NS蛋白进行研究 在不同的比例下，并分析形成的络合物。互动 将通过评估N蛋白与病毒RNA的能力来研究 表达的N蛋白对体外合成狂犬病病毒的包埋作用 引导RNA，从而保护它免受核糖核酸酶的消化。这个 狂犬病病毒N蛋白与NS蛋白及N蛋白之间的相互作用 而基因组RNA也将通过同时表达 一步法体外新药中的成分 转录/翻译系统。这些组件的区域(N 蛋白质、NS蛋白、基因组RNA)参与相互作用 也将通过表达带有C末端的蛋白突变体来鉴定 截断和内部缺失，并研究其结合活性 这些诱变的蛋白质。