Assembly of the Negative Stranded RNA Virus Core

非节段负链RNA病毒具有一个独特的特征，即其模板RNA总是被一个 核衣壳蛋白N.含有L蛋白和P蛋白的病毒聚合酶可以识别模板， 只有当基因组RNA与N.在前一时期， N-RNA复合物的结构和P的中心结构域的结构已被解决。功能推论来自 该结构用于制定本建议中的实验。该提案提出了四个具体目标： 目标1. P-N-RNA复合物的结构。我们的初步分析表明，有一个显着的构象 当P附着时，N-RNA复合物发生变化。这可能意味着P诱导了N-RNA的变化， 模板，其是由含L的聚合酶识别所需的。复杂的结构将被确定 结合冷冻电镜和X射线晶体学EM结构将提供一个框架， 可以构建单个P结构域和N-RNA复合物的结构。目标2.解析P和N的相互作用。 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 <$-P复合物被募集到复制位点，然后N <$被 组装成新生的N-RNA链。在这个目标中，N将被修剪和突变，因此它不会使RNA发生糖苷化 在P或其片段存在下也不形成大的寡聚体。此外，P的功能与复制有关， 目的3.建立一种新的反向遗传系统，以确定其转录水平。复制酶复合物形成的研究 正如Banerjee小组提出的，VSV复制酶复合物由L、N和P形成。 构建了组成型共表达L、N和P的细胞系。为了研究这种复制酶复合体的功能， 在结构上，我们将纯化足够量的复合物。将确定每种组分的化学计量 并且复合物将经受cryoEM研究和结晶。从L、N或 P将在新的反向遗传系统中进行测试。目标4。M-P-N-RNA复合物的结构。述基质蛋白 VSV组装需要M。然而，其在这一进程中的作用没有明确界定。从我们以前的P-N扩展 在共表达构建体中，我们已经产生了共表达M、P和N的构建体。将M与P-N-聚乙二醇共纯化， RNA复合物。该复合物的结晶和冷冻EM研究正在进行中。结果将显示M如何可以 与N-RNA复合物相互作用，可能也与P相互作用。

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.