Assembly of the Negative Stranded RNA Virus Core

负链RNA病毒核心的组装

• 批准号: 7880321
• 负责人: MING LUO
• 金额: $ 1.82万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-14 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7880321
• 关键词: Biological; Cell Line; Complex; Cryoelectron Microscopy; Crystallization; Data; Dissection; Elements; Genetic Transcription; Genome; Genomics; Glycine decarboxylase; Individual; L Forms; Mutate; Nucleocapsid Proteins; Phosphoproteins; Play; Polymerase; Process; Proteins; RNA; RNA Binding; RNA Viruses; Recruitment Activity; Roentgen Rays; Role; Series; Shapes; Site; Structure; System; Testing; Trefoil Motif; Vesicular stomatitis Indiana virus; Viral; Virion; X-Ray Crystallography; base; design; interest; multiple myeloma M Protein; novel; positional cloning; prevent; replicase; research study; stoichiometry; three dimensional structure; viral RNA; virus core

Nonsegmented negative strand RNA viruses have a unique feature that its template RNA is always enwrapped by a nucleocapsid protein N. The viral polymerase containing L and P proteins could recognize the template during transcription and replication only when the genomic RNA is associated with N. In the previous period, the crystal structure of a N-RNA complex and that of the central domain of P have been solved. Functional inferences derived from the structure are used in formulating the experiments in this proposal. Four specific aims are presented in this proposal: Aim 1. Structure of the P-N-RNA complex. Our initial analysis indicated that there is a significant conformational change in the N-RNA complex when P is attached. The implication may be that P induces a change in the N-RNA template that is required for the recognition by the L containing polymerase. The complex structure will be determined by combination of cryoEM and X-ray crystallography. The EM structure will provide the framework in which the atomic structures of individual P domains and the N-RNA complex could be built. Aim 2. Dissection of P and N interaction. Based on the three dimensional structures of N and P, we have designed a series of experiments to define the interactions of P with N¿ to reveal their functions. The N¿-P complex is recruited into the site of replication and N¿ is then assembled into the nascent N-RNA strand. In this aim, N will be trimmed and mutated so it will not encapsidate RNA nor form large oligomers in the presence of P or its fragments. Further more, functions of P related to replication versus transcription will be determined with a novel reverse genetic system.Aim 3. Study of the replicase complex formed by L, N and P. As proposed by Banerjee's group, the VSV replicase complex is formed by L, N and P. We have constructed a cell line that co-expresses L, N and P constitutively. To study this replicase complex functionally and structurally, we will purify the complex in sufficient quantities. The stoichiometry of each component will be determined and the complex will be subject to cryoEM studies and crystallization. Functional inferences from the structure of L, N or P will be tested in the novel reverse genetic system. Aim 4. Structure of the M-P-N-RNA complex. The matrix protein M is required for VSV assembly. However, its role in the process is not clearly defined. Extended from our previous P-N coexpression construct, we have generated a construct that co-expresses M, P and N. M was copurified with the P-N- RNA complex. Crystallization and cryoEM studies of this complex are in progress. The result will show how M may interact with the N-RNA complex, and perhaps with P as well.

非分段负链RNA病毒有一个独特的特征，它的模板RNA总是被一个 核衣壳蛋白N含有L和P蛋白的病毒聚合酶在 只有当基因组RNA与氮相关时才能转录和复制。在前一时期，晶体 解决了N-RNA复合体的结构和P的中心结构域的结构问题。函数推论源自 该结构用于制定本方案中的实验。这项提案提出了四个具体目标： 目的1.P-N-RNA复合体的结构。我们的初步分析表明，有一个重要的构象 当P附着时，N-RNA复合体的变化。这可能意味着磷诱导了N-RNA的变化 含有聚合酶的L识别所需的模板。复杂的结构将被确定 采用低温电子显微镜和X射线结晶学相结合的方法。EM结构将提供一个框架，其中原子 可以构建单个P结构域和N-RNA复合体的结构。目的2.剖析磷与氮的交互作用。 基于N和P的三维结构，我们设计了一系列实验来确定 P与N的相互作用以揭示其功能。N？-P复合体被招募到复制位置，然后N？ 组装成新生的N-RNA链。在这个目标中，N将被修剪和突变，这样它就不会包裹RNA 在P或其片段存在的情况下也不形成大的低聚物。此外，P与复制相关的功能与 转录将用一种新的反向遗传系统来确定。目的3.复制酶复合体形成的研究 由L、N和P提出，VSV复制酶复合体是由L、N和P形成的。 构建了结构性共表达L、N、P的细胞系。为了从功能上研究这种复制酶复合体， 在结构上，我们将提纯足够数量的复合体。将确定每种成分的化学计量比 该复合体将接受低温EM研究和结晶。L、N或结构中的功能推论 P将在新的反向遗传系统中进行测试。目的4.M-P-N-RNA复合体的结构。基质蛋白 VSV装配需要M。然而，它在这一过程中的作用并未得到明确界定。从我们之前的P-N扩展 共表达构建，我们已经产生了一个共表达M、P和N的构建体。 RNA复合体。该复合体的结晶和低温电子显微镜研究正在进行中。结果将显示M如何 与N-RNA复合体相互作用，也可能与P相互作用。