RNA binding and packaging by retroviral Gag proteins

逆转录病毒 Gag 蛋白的 RNA 结合和包装

基本信息

批准号：
10034983
负责人：
Karin M Musier-Forsyth
金额：
$ 51.53万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-17 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10034983
关键词：
5&apos Untranslated Regions Acylation Adopted Affect Binding Binding Sites Biological Assay Biology Cell Culture Techniques Cells Characteristics Clinical Computer Models Data Drug Targeting Elements Ensure Fluorescence Resonance Energy Transfer Genetic Materials HIV-1 Heterogeneity Hydroxyl Radical In Vitro Kinetics Label Mass Spectrum Analysis Measures Mediating Molecular Conformation Mutation Nucleotides Pharmaceutical Preparations Primer Extension Production Property RNA RNA Binding RNA Conformation RNA Probes RNA Splicing Reporting Role Specificity Structure Testing Therapeutic Therapeutic Agents Transcript Transcription Initiation Site Viral Viral Genome Virion Virus Assembly Virus Replication Work Zinc Fingers base cost crosslink design dimer dimethyl sulfate expectation fitness gag Gene Products genomic RNA hammerhead ribozyme inhibitor/antagonist insight mutant novel strategies novel therapeutics particle preference prevent single molecule stoichiometry targeted treatment viral RNA

项目摘要

Abstract HIV-1 packages two copies of genomic RNA (gRNA) as a dimer into newly formed viral particles. Retroviral assembly doesn’t depend on the presence of gRNA, yet gRNA packaging is essential for production of infectious virus particles. Robust and specific mechanisms must therefore be in place to ensure the genetic material is passed on to progeny virions. While there are numerous therapeutics in clinical use, there are currently no gRNA packaging or viral assembly inhibitors. Selective gRNA packaging is facilitated by interactions between the HIV- 1 Gag protein and conserved gRNA elements within the 5′ untranslated region (5′UTR). Here, we focus on recent unexpected findings in HIV-1 RNA biology that revealed sequence heterogeneity at the 5′ end of the HIV-1 RNA transcript. The variable number of G residues (1G, 2G and 3G) has been reported to affect the gRNA localization, with 1G RNA preferentially selected over 3G to be the viral genome. This is very surprising given that these two 9-kb RNAs only differ by 2 nucleotides. Preliminary data presented here strongly support the enrichment of 1G- containing transcripts among packaged gRNA. Importantly, various point mutants that abrogate the preference for 1G RNA have been identified. Exciting preliminary data support our major hypothesis that the ensemble of RNA structures adopted by the 5′UTR is significantly altered in 1G vs. 3G transcripts. These data provide a strong starting point for the proposed studies aimed at elucidating the mechanism by which transcriptional start site choice modulates gRNA packaging selectivity. More broadly, we will gain insights into how subtle sequence changes can alter the ensemble of 5′UTR RNA structures and impact viral replication fitness. The specific aims are: (1) To probe wild-type and mutant retroviral gRNA structure and dynamics; (2) To probe wild-type and mutant HIV-1 Gag RNA binding and packaging specificity. The results of these studies will help guide the design of novel therapeutic agents that target the 5′UTR and interfere with the essential conformational plasticity and/or key binding interactions with the expectation for a lower rate of mutational escape than conventional drugs.

抽象的 HIV-1 将两个基因组 RNA (gRNA) 拷贝作为二聚体包装到新形成的逆转录病毒颗粒中。组装不依赖于 gRNA 的存在，但 gRNA 包装对于感染性病毒的生产至关重要因此，必须建立强大且特定的机制来确保遗传物质的存在。虽然临床上有多种治疗方法，但目前还没有 gRNA。 HIV-包装或病毒组装抑制剂之间的相互作用促进了选择性gRNA包装。 1 Gag 蛋白和 5' 非翻译区 (5'UTR) 内的保守 gRNA 元件在这里，我们重点关注最近的研究。 HIV-1 RNA 生物学中的意外发现揭示了 HIV-1 RNA 5' 端的序列异质性据报道，G 残基（1G、2G 和 3G）的可变数量会影响 gRNA 定位，考虑到这两个RNA，1G RNA优先选择而不是3G作为病毒基因组。 9-kb RNA 仅相差 2 个核苷酸，此处提供的初步数据强烈支持 1G- 的富集。重要的是，在包装的 gRNA 中包含转录本，各种点突变消除了偏好。 1G RNA 的令人兴奋的初步数据支持了我们的主要假设：与 3G 转录本相比，5'UTR 采用的 RNA 结构发生了显着改变。旨在阐明转录起始机制的拟议研究的有力起点更广泛地说，我们将深入了解序列的微妙程度。变化可以改变 5'UTR RNA 结构的整体并影响病毒复制的适应性。是： (1) 探测野生型和突变型逆转录病毒 gRNA 结构和动力学； (2) 探测野生型和突变型逆转录病毒 gRNA 结构和动力学；突变体HIV-1 Gag RNA结合和包装特异性这些研究的结果将有助于指导设计。靶向 5'UTR 并干扰基本构象可塑性和/或关键结合相互作用，预计突变逃逸率比传统药物更低。