RNA binding and packaging by retroviral Gag proteins

逆转录病毒 Gag 蛋白的 RNA 结合和包装

• 批准号: 10347332
• 负责人: Karin M Musier-Forsyth
• 金额: $ 48.38万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 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/10347332
• 关键词: 5' 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; insight; mutant; novel strategies; novel therapeutics; particle; preference; prevent; single molecule; stoichiometry; targeted treatment; viral RNA

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包装两份基因组RNA（GRNA）作为二聚体新形成的病毒颗粒。逆转录病毒 组装不取决于GRNA的存在，但GRNA包装对于产生感染性至关重要 病毒颗粒。因此，必须建立强大和特定的机制，以确保遗传物质是 传递到进展病毒。虽然临床使用中有许多治疗剂，但目前没有GRNA 包装或病毒组装抑制剂。选择性GRNA包装受HIV-之间的相互作用支持 1个GAG蛋白和在5'未翻译区域（5'UTR）中配置的GRNA元素。在这里，我们专注于最近 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 成绩单。据报道，g残基数（1g，2g和3g）的可变数量会影响GRNA定位， 1G RNA优先选择3G作为病毒基因组。鉴于这两个 9-kb RNA仅通过2个核苷酸而有所不同。此处提供的初步数据强烈支持1G-的富集 包含包装的GRNA中的转录本。重要的是，废除偏好的各种点突变体 对于1G RNA已被鉴定。令人兴奋的初步数据支持我们的主要假设 5'UTR采用的RNA结构在1G和3G转录本中显着改变。这些数据提供了 旨在阐明转录开始的机制的拟议研究的强大起点 站点选择调制GRNA包装选择性。更广泛地说，我们将深入了解微妙的顺序 变化可以改变5'UTR RNA结构的合奏并影响病毒复制适应性。具体目标 为：（1）探测野生型和突变逆转录病毒的结构和动力学； （2）探测野生型和 突变的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 针对5'UTR并干扰基本构象可塑性和/或干扰的新型治疗剂 与常规药物相比，与对突变逃逸率较低的期望的关键结合相互作用。