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IDENTIFICATION AND CHARACTERIZATION OF RNA STRUCTURES IN THE GENOME OF INFLUENZA VIRUS

流感病毒基因组 RNA 结构的鉴定和表征

基本信息

批准号：
10207357
负责人：
Adrianus CM Boon
金额：
$ 52.19万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-13 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10207357
关键词：
Address Affect Attenuated Avian Influenza A Virus Binding Biochemical Birds Cells Complex Coupled Defect Disease Elements Evolution Generations Genes Genetic Genome Goals Growth Health Human Image Analysis Immunoprecipitation In Vitro Individual Influenza Influenza A virus Lead Ligands Location Mediating Molecular Molecular Conformation Mutation Nature Nucleoproteins Nucleotides Polymerase Positioning Attribute Process Proteins RNA RNA Binding Ribonucleoproteins Risk Scientist Spanish flu Structure Testing Vaccines Variant Viral Viral Genome Viral Packaging Virion Virus Virus Replication attenuation crosslink crosslinking and immunoprecipitation sequencing driving force genome-wide in vivo influenzavirus insight multidisciplinary mutant new technology next generation sequencing novel pandemic influenza particle physical property preservation prevent structural biology viral RNA virology

项目摘要

PROJECT SUMMARY Influenza poses a significant health risk worldwide. It is estimated that between 250,000 and 500,000 people die each year from the virus. This number can increase dramatically during an influenza pandemic, such as the 1918 Spanish influenza pandemic, when more than 25 million people died. The genome of influenza A virus consists of eight segments of single-stranded, negative-sense RNA that are encapsidated as individual ribonucleoprotein complexes (RNPs). Each RNP contains one strand of RNA, a viral polymerase complex and multiple copies of the viral nucleoprotein (NP). The segmented nature of influenza virus allows for reassortment or mixing of gene-segment and this is a major contributor to the emergence of pandemic influenza viruses. Image analysis of individual RNP complexes show a double-helical conformation of two strands of NP connected by a small loop and the polymerase proteins at the termini of the complex. The organization of the viral RNA in this structure is not known, which limits our ability to understand and to target RNA dependent processes that contribute to the generation of pandemic influenza viruses. To address this gap, we applied a new technology, cross-linking and immunoprecipitation coupled with next-generation sequencing (CLIP-seq), to discern the organization of the RNA in native RNP complexes of IAV. Analysis of the immunoprecipitated viral RNA revealed non-uniform binding of the NP to the genome. Specifically, we identified 34 regions that range in size between 14-85 nucleotides that are significantly underrepresented in the NP-bound RNA fraction. Nearly 50% of the low-NP binding regions are predicted to form stable secondary structures, including RNA hairpins and RNA pseudoknot structures. This led us to hypothesize that the RNA structural features, that are poorly bound by NP, are pivotal for gene-segment interactions and are the primary features controlling IAV segment packaging at the molecular level. In support of this hypothesis we found that the introduction of synonymous structural mutations that disrupt the predicted RNA structures attenuated the resulting viruses without exception. In contrast, synonymous mutations that did not alter the predicted RNA structure, or mutations in control regions had no impact on the growth of the virus. The attenuation in low-NP binding mutant viruses was due to a defect in genome packaging. The goal of this application is to determine the mechanism by which low-NP binding regions affect IAV replication, genome packaging, and virus reassortment.

项目摘要流感在全球范围内构成重大健康风险。据估计，25万至50万人每年死于病毒。在流感大流行期间，这一数字可能会急剧增加， 1918年西班牙流感大流行，超过2500万人死亡。甲型流感病毒的基因组由8段单链、反义RNA组成，核糖核蛋白复合物（RNP）。每个RNP含有一条RNA链，一种病毒聚合酶复合物，病毒核蛋白（NP）的多个拷贝。流感病毒的分段性质允许基因片段的重组或混合，这是大流行病出现的主要原因。流感病毒。单个RNP复合物的图像分析显示两个RNP复合物的双螺旋构象。由小环连接的NP链和复合物末端的聚合酶蛋白。的这种结构中病毒RNA的组织尚不清楚，这限制了我们理解和靶向的能力。 RNA依赖的过程，有助于大流行性流感病毒的产生。为了解决这个 gap，我们应用了一种新的技术，交联和免疫沉淀结合下一代测序（CLIP-seq），以辨别IAV的天然RNP复合物中RNA的组织。分析免疫沉淀的病毒RNA显示NP与基因组的非均匀结合。我们特别鉴定了34个区域，其大小范围在14-85个核苷酸之间，这些区域在基因组中明显代表不足。 NP结合的RNA部分。近50%的低NP结合区域被预测形成稳定的二级结构结构，包括RNA发夹和RNA假结结构。这让我们假设RNA 与NP结合不好的结构特征是基因片段相互作用的关键，也是主要的特征在于在分子水平上控制IAV片段包装。为了支持这一假设，我们发现，引入破坏预测RNA结构的同义结构突变，病毒无一例外。相反，没有改变预测的RNA的同义突变，结构或控制区的突变对病毒的生长没有影响。低NP中的衰减结合突变病毒是由于基因组包装的缺陷。此应用程序的目标是确定低NP结合区影响IAV复制、基因组包装和病毒的机制重组