Transcription and replication in nonsegmented negative-strand RNA viruses

 描述(申请人提供)：负链RNA病毒(NSV)包括麻疹、甲型流感、埃博拉和许多其他严重人类疾病的病原体。为了更好地了解这类病毒的复制周期，我们正在研究水疱性口炎病毒(VSV)的分子结构，水疱性口炎病毒是横纹病毒家族中一种典型的非节段性NSV(NNSV)。VSV和另一种NNSV编码一组功能保守的蛋白质(N、P和L)，这些蛋白质用于复制它们的基因组并产生功能性病毒mRNAs。转录和复制过程需要两个病毒成分，依赖核糖核酸的核糖核酸聚合酶(RdRp，L和P之间的复合体)和基因组模板。对于NSV，这两个过程的活性模板都是核衣壳(N+RNA)，而不仅仅是裸露的RNA。我们以前的结构研究表明，RNA是如何被核衣壳包裹的，以及一旦进入衣壳，RNA是如何采用独特的结构的。RdRp必须接触到被隔离的RNA的碱基，才能启动多核苷酸合成。由于核衣壳是病毒RNA合成的模板，并且N与RNA之间存在密切的联系，因此N蛋白在转录和复制中可能发挥什么作用的问题出现了。本文提出的新研究主要集中在：目的1，病毒转录和复制启动过程中功能模板的结构要求。我们已经开发了一些方法来解决包裹在核衣壳样粒子(NLP)中的特定RNA序列的结构。为此，我们计划解决一系列新的结构，以解决N蛋白如何帮助RdRp识别核衣壳内的病毒特异性RNA序列(如3‘基因组末端和转录起始序列)的问题。除了这些研究，还将研究影响转录和复制的N蛋白的结构变化。这将通过研究一系列具有影响这些酶过程的表型的突变N蛋白来实现。这些突变提示N蛋白本身在转录和复制调控中起作用。在目标2中，我们将从L和P蛋白的作用来研究多核苷酸的合成。我们已经设计了一系列L蛋白质片段，这些片段是可溶的。N-端区和C-端区已经结晶，N-端区的衍射率达到&gt；2.8？分辨率。我们将确定L的几个结构域的结构。 与L、P和N蛋白的EM研究相结合，旨在重建更大的三方复制酶复合体和核衣壳。总的来说，这里提出的研究将从两个角度讨论NSV的复制和转录，模板以及参与这些关键酶过程的机制。我们的研究结果可能会产生一些信息，这些信息将促进未来针对这类病原体的药物设计策略。

 DESCRIPTION (provided by applicant): Negative-strand RNA viruses (NSVs) include measles, influenza A, Ebola and many other causative agents of serious human disease. In order to gain a better understanding of the replication cycle of this class of viruses, we are studying the molecular structure of vesicular stomatitis virus (VSV), a prototypic non-segmented NSV (NNSV) in the rhabdovirus family. VSV and the other NNSV encode a functionally conserved set of proteins (N, P, and L) that serve to duplicate their genomes and produce functional viral mRNAs. The processes of transcription and replication require two viral components, the RNA-dependent RNA polymerase (RdRp, a complex between L and P) and the genomic template. For NSVs, the active template for both of these processes is the nucleocapsid (N+RNA), not the naked RNA alone. Our previous structural studies showed how the RNA is encapsidated by the nucleocapsid and how the RNA adopts a unique structure once within the capsid. The RdRp must gain access to the bases of the sequestered RNA in order to initiate polynucleotide synthesis. Since the nucleocapsid is the template for viral RNA synthesis and given the intimate association between N and the RNA, questions arise as to what role the N protein may play in transcription and replication. The new studies proposed here are focused on: Aim 1, the structural requirement of the functional template in initiation of viral transcriptin and replication. We have developed methods to solve the structure of specific RNA sequences encapsidated within nucleocapsid-like particles (NLPs). In this aim, we plan to solve a novel series of structures that address the question about how the N protein helps the RdRp to recognize viral specific RNA sequences (such as the 3' genomic termini and transcriptional initiation sequences) within the nucleocapsid. These studies will be complemented with a look at structural changes in the N protein that affect transcription and replication. This will be accomplished by studying a series of mutant N proteins with phenotypes that affect these enzymatic processes. These mutants suggest that the N protein itself plays a role in regulation of transcription and replication. In Aim 2, we will examine polynucleotide synthesis from the role of the L and P proteins. We have engineered a series of L protein fragments that are soluble. The N- and C-terminal regions have already been crystallized with the N-terminal domain diffracting to >2.8Å resolution. We will determine structures for several domains of L. These will be integrated with EM studies of the L, P and N proteins aimed at reconstructing the larger tripartite replicase complex and nucleocapsids. Collectively, the studies proposed here will address both replication and transcription of NSV from two perspectives, the template as well as the machinery involved in these essential enzymatic processes. The outcome of our studies may yield information that will promote future drug design strategies against this group of pathogens.