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: 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
• 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' 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

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.

摘要 HIV-1将两个拷贝的基因组RNA（gRNA）作为二聚体包装成新形成的病毒颗粒。录病毒 组装不依赖于gRNA的存在，但gRNA包装对于产生感染性的 病毒颗粒因此，必须建立强有力的具体机制，以确保遗传物质 传递给后代病毒体。虽然临床上有许多治疗方法，但目前还没有gRNA 包装或病毒组装抑制剂。选择性gRNA包装是通过HIV- 1 Gag蛋白和5′非翻译区（5′UTR）内的保守gRNA元件。在这里，我们关注最近的 HIV-1 RNA生物学中的一项意外发现，揭示了HIV-1 RNA 5′端的序列异质性 成绩单。已经报道了可变数目的G残基（1G、2G和3G）影响gRNA定位， 其中1G RNA优先于3G被选择为病毒基因组。令人惊讶的是，这两个 9-kb RNA仅相差2个核苷酸。这里提供的初步数据强烈支持1G的富集- 包含包装的gRNA中的转录物。重要的是，各种取消偏好的点突变体 1G RNA的表达。令人兴奋的初步数据支持我们的主要假设， 5 'UTR所采用的RNA结构在1G与3G转录物中显著改变。这些数据提供了一个 强有力的起点，为拟议的研究，旨在阐明机制，转录启动 位点选择调节gRNA包装选择性。更广泛地说，我们将深入了解如何微妙的序列 变化可以改变5 'UTR RNA结构的整体并影响病毒复制适应性。具体目标 目的是：（1）探测野生型和突变型逆转录病毒gRNA的结构和动力学;（2）探测野生型和突变型逆转录病毒gRNA的结构和动力学。 突变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的新型治疗剂和/或 关键的结合相互作用，预期突变逃逸率低于常规药物。