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.

项目摘要流感在世界范围内带来了重大健康风险。据估计，250,000至500,000人每年死于病毒。在流感大流行期间，这个数字可以大大增加 1918年，西班牙流感大流行，当时有超过2500万人死亡。流感病毒的基因组由八个段的单链，负义的RNA组成，它们被封装为个体核糖核蛋白复合物（RNP）。每个RNP包含一条RNA，一个病毒聚合酶复合酶，病毒核蛋白（NP）的多个副本。流感病毒的分段性质允许重新分类或基因段的混合，这是大流行的主要原因流感病毒。单个RNP复合物的图像分析显示了两个的双螺旋构象通过小环和复合物末端的聚合酶蛋白连接的NP的链。这该结构中病毒RNA的组织尚不清楚，这限制了我们理解和靶向的能力 RNA依赖性过程有助于产生大流行性流感病毒。解决这个问题差距，我们应用了一项新技术，交联和免疫沉淀以及下一代测序（夹子seq），以辨别IAV天然RNP复合物中RNA的组织。分析免疫沉淀的病毒RNA揭示了NP与基因组的不均匀结合。具体来说，我们确定了34个区域，大小范围在14-85个核苷酸之间，这些区域在 NP结合的RNA分数。预计将近50％的低NP结合区域形成稳定的次级结构，包括RNA发夹和RNA伪诺结构。这使我们假设RNA 由NP约束的结构特征对于基因段相互作用是关键的，并且是主要的在分子水平上控制IAV段包装的特征。为了支持这一假设，我们发现引入的同义结构突变破坏了预测的RNA结构使无一例外的导致病毒。相反，没有改变预测的RNA的同义突变结构或对照区域的突变对病毒的生长没有影响。低NP的衰减结合突变病毒是由于基因组包装缺陷所致。该应用的目的是确定低NP结合区域影响IAV复制，基因组包装和病毒的机制重新分类。