Transcription and replication in nonsegmented negative-strand RNA viruses

非节段负链RNA病毒的转录和复制

基本信息

批准号：
9195690
负责人：
Todd Jason Green
金额：
$ 36.75万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-01 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9195690
关键词：
Address Adopted Affect Amino Acid Sequence Biochemical Genetics C-terminal Capsid Complement Complex Crystallization Data Data Analyses Docking Drug Design Ebola virus Electron Microscopy Engineering Exhibits Family Future Genes Genetic Transcription Genetic study Genome Genomics Glycine decarboxylase Goals Hybrids Influenza A virus Knowledge Length Measles Messenger RNA Methods Modeling Molecular Structure Mutation Nucleocapsid Nucleocapsid Proteins Outcome Phenotype Play Poly A Polymerase Polynucleotides Process Protein Fragment Proteins Publishing RNA RNA Sequences RNA Viruses RNA chemical synthesis RNA-Directed RNA Polymerase Replication-Associated Process Resolution Rhabdoviridae Roentgen Rays Role Series Set protein Site Structure Transcription Initiation Transcription Process Transcriptional Regulation Vesicular stomatitis Indiana virus Viral Viral Proteins Virus Virus Replication X-Ray Crystallography base experimental study genomic RNA human disease insight member mutant novel particle pathogen protein function public health relevance replicase reverse genetics transcription termination vesicular stomatitis virus L protein viral RNA

项目摘要

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.

描述（由适用提供）：负链RNA病毒（NSV）包括麻疹，影响力，埃博拉病毒和许多其他严重人类疾病的严重药物。为了更好地了解这类病毒的复制周期，我们正在研究色托病毒家族中原型非细分NSV（NNSV）的囊泡口腔炎病毒（VSV）的分子结构。 VSV和其他NNSV编码功能构成的蛋白质集（N，P和L），这些蛋白质（N，P和L）用于复制其基因组并产生功能性病毒mRNA。转录和复制过程需要两个病毒成分，即RNA依赖性RNA聚合酶（RDRP，L和P之间的复合物）和基因组模板。对于NSV，这两个过程的主动模板是核Ocapsid（N+RNA），而不是单独的裸RNA。我们以前的结构研究表明，RNA是如何被核ocapsid封装的，以及RNA如何在衣壳内采用独特的结构。 RDRP必须访问隔离的RNA的碱基，以启动多核苷酸合成。由于核OCAPSID是病毒RNA合成的模板，并且鉴于N和RNA之间的亲密关联，因此就N蛋白在转录和复制中起着什么作用提出了问题。此处提出的新研究集中在：AIM 1，功能模板的结构要求启动病毒转录蛋白和复制时。我们已经开发了解决封装在核Ocapsid样颗粒（NLP）中的特定RNA序列的结构的方法。在此目标中，我们计划解决一系列新的结构，以解决有关N蛋白如何帮助RDRP识别核Ocapsid中病毒特异性RNA序列（例如3'基因组末端和转录起始序列）的问题。这些研究将完成，以查看影响转录和复制的N蛋白的结构变化。这将通过研究一系列具有影响这些酶促过程的表型的突变N蛋白来实现。这些突变体认为N蛋白本身在调节转录和复制中起作用。在AIM 2中，我们将从L和P蛋白的作用中检查多核苷酸的合成。我们已经设计了一系列固体的L蛋白片段。 N末端区域和C末端区域已经通过N末端结构域衍射到>2.8Å分辨率结晶。我们将确定L的多个域的结构。与旨在重建较大三方复制酶复合物和核蛋白质的L，P和N蛋白的EM研究相结合。总的来说，此处提出的研究将从两个角度来解决NSV的复制和转录，分别是模板以及这些基本酶促过程所涉及的机械。我们研究的结果可能会产生信息，以促进对这组病原体的未来药物设计策略。