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

项目总结 流感在全球范围内构成重大的健康风险。据估计，有25万到50万人 每年都会死于这种病毒。在流感大流行期间，这一数字可能会急剧增加，例如 1918年西班牙流感大流行，当时有超过2500万人死亡。甲型流感病毒基因组 由八段单链负义RNA组成，这些片段被封装为单独的 核糖核蛋白复合体(RNP)。每个RNP含有一条RNA链、一个病毒聚合酶复合体和 病毒核蛋白(NP)的多个副本。流感病毒的节段性允许 基因片段的重新分类或混合，这是大流行出现的主要原因 流感病毒。单个RNP复合体的图像分析显示出两个双螺旋构象 由小环连接的NP链和位于复合体末端的聚合酶蛋白。这个 病毒RNA在这种结构中的组织尚不清楚，这限制了我们理解和靶向的能力 导致大流行性流感病毒产生的依赖RNA的过程。要解决这个问题 GAP，我们应用了一项新技术，交联和免疫沉淀与下一代 测序(CLIP-SEQ)，以辨别IAV天然RNP复合体中RNA的组织结构。分析 免疫沉淀的病毒RNA显示NP与基因组的结合不均匀。具体来说，我们 确定了34个大小在14-85个核苷酸之间的区域，这些区域在 NP结合的RNA组分。近50%的低NP结合区预计会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 不受NP结合的结构特征是基因-片段相互作用的关键，也是主要的 在分子水平上控制IAV节段包装的功能。为了支持这一假设，我们发现 同义结构突变的引入破坏了预测的RNA结构，削弱了 由此产生的病毒无一例外。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 重新编排。

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.