Structural basis of vesucular stomatitis virus transcription and replication

描述(申请人提供)：负链RNA病毒(NRVS)，如水疱性口炎病毒(VSV)，是独特的，因为它们的核衣壳，而不是裸露的RNA，是转录和复制的活性模板。在整个感染周期中，基因组RNA被核衣壳蛋白(N)完全隔离。病毒核糖核酸依赖的核糖核酸聚合酶(RdRp)是L和P之间的复合体，必须接触到核糖核酸的碱基才能启动转录或复制。我们之前的结构研究 展示了RNA是如何被核衣壳包裹的，以及RNA在包封腔内的结构和可及性。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，功能模板在病毒转录和复制启动过程中的结构要求。我们已经开发出了解决结构问题的方法 包埋在类核衣壳颗粒(NLP)中的特定RNA序列。在这个目标中，我们计划解决一系列新的结构，旨在解决N蛋白如何帮助RdRp识别隔离在核衣壳中的特定RNA序列的问题。此外，还将研究影响转录/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。这些突变表明N蛋白本身在转录/复制调控中起作用。在目标2中，我们将从L和P蛋白的作用而不是功能模板来研究多核苷酸的合成。我们已经设计了一系列L的可溶性蛋白质片段，其中两个已经结晶。我们将确定L的几个结构域的结构，这些结构将与旨在重建更大的三方复制酶复合体的L、P和N蛋白的EM研究相结合。总的来说，这里提出的研究将解决这两个复制问题 和转录从两个角度，模板以及参与这一关键的酶过程的机制。

DESCRIPTION (provided by applicant): Negative strand RNA viruses (NRVs), such as vesicular stomatitis virus (VSV), are unique because their nucleocapsid, not the naked RNA, is the active template for transcription and replication. During the complete infection cycle, the genomic RNA is completely sequestered by the nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRp, a complex between L and P) must gain access to the bases of the RNA in order to initiate transcription or replication. Our previous structural studies showed how the RNA is encapsidated by the nucleocapsid, as well as the structure and accessibility of the RNA within the encapsidation cavity. 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 transcription 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 aimed toward addressing the question about how the N protein helps RdRp to recognize specific RNA sequences sequestered in the nucleocapsid. This will be complemented with a look at structural changes in the N protein that affect transcription/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/replication. In Aim 2, we will examine polynucleotide synthesis from the role of the L and P proteins, rather than the functional template. We have engineered a series of L protein fragments that are soluble, two of which have been crystallized. 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. Collectively, the studies proposed here will address both replication and transcription from two perspectives, the template as well as the machinery involved in this critical enzymatic process.