FLU VIRUS RNA TRANSCRIPTION MACHINE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人副流感病毒3型(HPIV3)是一种包膜病毒，其基因组全长为15462个核苷酸(GenBank登录号：NC_001796)，共编码8个蛋白。基因组转录和复制的基本单位是核衣壳蛋白(N，515个残基)、磷蛋白(P，603个残基)和大蛋白(L，2233个残基)以及核糖核酸基因组。N蛋白与基因组或反基因组RNA结合，形成稳定的抗核糖核酸酶螺旋核衣壳。P蛋白含有一个寡聚化结构域，并被高度磷酸化。L蛋白是一种大型多功能蛋白质，负责催化核糖核酸的转录/复制。P蛋白在RNA转录/复制机制的组装中起着关键作用。除了能够自组装成同源低聚物外，P蛋白还可以与N和L结合，从而将L-P复合体连接到核衣壳上，完成机械的组装。 我们目前正在建立针对HPIV3的反向遗传学系统。一旦这样的系统可用，我们将能够标记每个单独的病毒蛋白，并单独或以复合体的形式纯化它们。还将从感染HPIV3的细胞中提纯核衣壳。我们已经在实验室建立了酵母和昆虫细胞表达系统，并将分别生产螺旋核衣壳和环状核衣壳。所有用于单粒子冷冻-EM分析的配合物均具有接近或超过200kD的分子量。我们相信，通过亲和层析和尺寸排除层析将获得高质量的纯化样品。洗涤剂和高盐浓度不太可能包含在最终缓冲液中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Human parainfluenza virus 3 (HPIV3) is an enveloped virus containing a negative-sense single-stranded RNA genome of 15462 nucleotides long (GenBank Accession No. NC_001796), encoding a total of 8 proteins. The basic unit for genome transcription and replication is composed of nucleocapsid protein (N, 515 residues), phosphoprotein (P, 603 residues) and large protein (L, 2233 residues) together with the RNA genome. The N protein associates with genomic or anti-genomic RNA to form a stable RNase-resistant helical nucleocapsid. The P protein contains an oligomerization domain and is heavily phosphorylated. The L protein is a large multifunctional protein responsible for the catalytic activities of RNA transcription/replication. The P protein plays a key role in assembly of the RNA transcription/replication machinery. In addition to be able to self-assemble into homo-oligomers, the P protein can also associate with N and L, thereby linking L-P complex to the nucleocapsid to complete the assembly of the machinery. We are currently in the process of establishing a reverse genetics system for HPIV3. Once such a system is available, we will be able to tag each individual viral protein and purify them individually or in complex. Purification of nucleocapsid from HPIV3-infected cells will also be carried out. We have already established yeast and insect cell expression systems in the lab, and will produce helical nucleocapsids or ring-like nucleocapsids respectively. All the proposed complexes for single-particle cryo-EM analysis have molecular weights close or over 200 kD. We are confident that high quality purified samples will be obtained through affinity and size-exclusion chromatography. Detergent and high-salt concentration are not likely to be contained in the final buffer.