Transcription and replication in nonsegmented negative-strand RNA viruses

非节段负链RNA病毒的转录和复制

• 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
• 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

 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（由申请方提供）：负链RNA病毒（NSV）包括麻疹、甲型流感、埃博拉病毒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，我们正在研究水泡性口炎病毒（VSV）的分子结构，这是弹状病毒家族中的一种原型非节段NSV（NNSV）。VSV和其他NNSV编码一组功能保守的蛋白质（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种病毒成分，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，这两个过程的活性模板是核衣壳（N+RNA），而不仅仅是裸RNA。我们以前的结构研究显示了RNA是如何被核衣壳包裹的，以及RNA一旦进入衣壳如何采用独特的结构。RdRp必须接近被隔离的RNA的碱基以启动多核苷酸合成。由于核衣壳是病毒RNA合成的模板，并且考虑到N和RNA之间的密切联系，因此出现了N蛋白在转录和复制中可能发挥什么作用的问题。本文提出的新研究集中在：目的1，功能模板在病毒转录和复制起始中的结构要求。我们已经开发了解决特定RNA序列的结构的方法，这些RNA序列在核衣壳样颗粒（NLP）中被包裹。在这个目标中，我们计划解决一系列新的结构，解决了关于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。这些研究将补充看N蛋白的结构变化，影响转录和复制。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。这些突变体表明N蛋白本身在转录和复制的调节中起作用。在目标2中，我们将从L和P蛋白的作用来研究多核苷酸合成。我们已经设计了一系列可溶性的L蛋白片段。N-和C-末端区域已经结晶，N-末端域衍射到> 2.8 nm分辨率。我们将确定L的几个域的结构。这些将 整合EM研究的L，P和N蛋白，旨在重建较大的三重复制酶复合物和核衣壳。总的来说，这里提出的研究将从两个角度来解决NSV的复制和转录，模板以及这些基本的酶促过程中所涉及的机器。我们的研究结果可能会产生信息，将促进未来的药物设计策略，对这组病原体。