RNA binding and packaging by retroviral Gag proteins

摘要 HIV-1将两个拷贝的基因组RNA(GRNA)作为二聚体包装到新形成的病毒颗粒中。逆转录病毒 组装不依赖于gRNA的存在，但gRNA包装对于产生感染性 病毒颗粒。因此，必须建立健全和具体的机制，以确保遗传物质 传给了子代病毒粒子。虽然有许多治疗方法在临床上使用，但目前还没有grna。 包装或病毒组装抑制剂。选择性的gRNA包装是由HIV- 1个Gag蛋白和5‘非翻译区(5’UTR)保守的gRNA元件。在这里，我们重点关注最近的 HIV-1RNA生物学中的意外发现，揭示了HIV-1RNA 5‘端的序列异质性 文字记录。据报道，可变数量的G残基(1G、2G和3G)会影响gRNA的定位， 1G RNA优先选择为病毒基因组，而不是3G。这非常令人惊讶，因为这两个人 9kb的RNA只有两个核苷酸的差异。这里提供的初步数据有力地支持了1G- 在包装的gRNA中包含转录本。重要的是，消除这种偏好的各种点突变 针对1G的RNA已被鉴定。令人兴奋的初步数据支持我们的主要假设，即 在1G和3G转录本中，5‘UTR采用的RNA结构发生了显著变化。这些数据提供了一个 为拟议的研究提供了强有力的起点，旨在阐明转录启动的机制 地点选择调节gRNA包装的选择性。更广泛地说，我们将深入了解微妙的序列 变化可以改变5‘UTRRNA结构的集合，并影响病毒复制的适合性。具体目标 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 突变的HIV-1 Gag RNA结合和包装的特异性。这些研究的结果将有助于指导设计 靶向5‘非编码区并干扰基本构象可塑性和/或 与期望比传统药物更低的突变逃逸率的关键结合作用。

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.