INTERACTIONS WITHIN RABIES VIRUS RNP COMPLEX

狂犬病病毒核糖核蛋白（RNP）复合物，由 被核蛋白（N）包围的基因组负链RNA， 磷蛋白（NS）和RNA聚合酶（L）能够启动 感染细胞，因此是必要的单位， 转录和复制。 N和NS蛋白是主要的蛋白质， RNP复合物的组成部分，并被认为在 病毒RNA通过相互作用进行转录和复制的过程 以及与L蛋白和病毒RNA的相互作用。 作为第一 进一步了解狂犬病病毒的转录， 复制，我建议研究狂犬病毒N蛋白的结构 及其与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株的蛋白质将在昆虫细胞中表达， 使用杆状病毒载体，并单独纯化或作为复合物纯化。 这些蛋白质或蛋白质复合物的详细结构将在下文中描述。 通过结晶和X射线衍射研究。 N的相互作用 将纯化的N蛋白和NS蛋白混合， 并分析所形成的复合物。 的相互作用 将通过评估N蛋白与病毒RNA结合的能力， 表达的N蛋白对体外合成的狂犬病病毒的抑制作用 前导RNA，从而保护其免受核糖核酸酶消化。 的 狂犬病毒N蛋白与NS蛋白相互作用及N蛋白与NS蛋白相互作用 和基因组RNA也将通过同时表达 成分在新的可用的一步体外 转录/翻译系统。 这些组件的区域（N 蛋白质、NS蛋白质、基因组RNA）参与相互作用 也将通过表达具有C-末端的蛋白质突变体来鉴定 截短和内部缺失，并研究的结合活性， 这些突变蛋白质。

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.