FLU VIRUS RNA TRANSCRIPTION MACHINE

流感病毒RNA转录机

• 批准号: 8168577
• 负责人: Rong Chen
• 金额: $ 0.65万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168577
• 关键词: Affinity; Buffers; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Detergents; Flu virus; Funding; Genbank; Genetic Transcription; Genome; Genomics; Glycine decarboxylase; Grant; Homo; Human Parainfluenza Virus 3; Individual; Insecta; Institution; Link; Molecular Sieve Chromatography; Molecular Weight; Nucleocapsid; Nucleocapsid Proteins; Nucleotides; Phosphoproteins; Play; Process; Proteins; RNA; Research; Research Personnel; Resistance; Resources; Ribonucleases; Role; Sampling; Source; System; United States National Institutes of Health; Viral Proteins; Virus; Yeasts; particle; positional cloning

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 人副流感病毒3型（HPIV3）是一种有包膜病毒，含有15462个核苷酸长的负义单链RNA基因组（GenBank登录号NC_001796），总共编码8种蛋白质。基因组转录和复制的基本单位由核衣壳蛋白（N，515个残基）、磷蛋白（P，603个残基）和大蛋白（L，2233个残基）以及RNA基因组组成。 N 蛋白与基因组或抗基因组 RNA 结合，形成稳定的抗 RNase 螺旋核衣壳。 P 蛋白含有寡聚结构域并且被高度磷酸化。 L蛋白是一种大型多功能蛋白，负责RNA转录/复制的催化活性。 P 蛋白在 RNA 转录/复制机制的组装中发挥着关键作用。 P蛋白除了能够自组装成同源寡聚体外，还可以与N和L缔合，从而将L-P复合物与核衣壳连接起来，完成机器的组装。 我们目前正在建立 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复合地纯化它们。还将对 HPIV3 感染细胞的核衣壳进行纯化。我们已经在实验室建立了酵母和昆虫细胞表达系统，将分别生产螺旋状核衣壳或环状核衣壳。所有提议的用于单粒子冷冻电镜分析的复合物的分子量接近或超过 200 kD。我们相信，通过亲和层析和尺寸排阻层析将获得高质量的纯化样品。最终缓冲液中不可能含有洗涤剂和高盐浓度。