Structural dynamics of the HIV-1 genomic 5' UTR

HIV-1 基因组 5 UTR 的结构动力学

• 批准号: 10102498
• 负责人: James B Munro
• 金额: $ 20.31万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-08 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10102498
• 关键词: Structural; dynamics; HIV; genomic; UTR

ABSTRACT The highly conserved 5' untranslated region (UTR) of the HIV-1 genome plays a central role in regulating viral replication. Groundbreaking NMR experiments, along with significant biochemical data support a model in which the 5'UTR can transition between at least two conformational states: in one state the genome remains a monomer, leading to translation of the viral genes; the second state is competent for dimerization and packaging into assembling virions. Therefore, according to this model the conformation of the 5'UTR determines the fate of each genome molecule. How the virus maintains an appropriate balance of genomes fated to packaging and translation remains unknown. But significant evidence indicates that viral proteins, as well as, potentially, host factors, facilitate switching between these functional states of the 5'UTR. The viral Gag protein in particular contains two RNA-binding domains, the nucleocapsid (NC) and matrix (MA) domains. Efficient recruitment of the dimerized genome into assembling virions occurs by way of specific interactions between the NC domain of Gag and the 5'UTR. As a nucleic acid chaperone, NC facilitates folding of the genome into thermodynamically favorable conformations that likely favor dimerization and packaging. In contrast, the MA domain counteracts the activity of NC either through interaction with NC or by modulating the structure of the 5'UTR. We have established a single-molecule Förster resonance energy transfer (smFRET) imaging approach to visualize the conformational dynamics of the 5'UTR. In our approach individual UTR molecules carrying donor and acceptor fluorophores are surface immobilized and imaged with total internal reflection fluorescence (TIRF) microscopy. Here, we will further develop this approach to elucidate the order and timing of 5'UTR conformational changes, dimerization, and Gag binding events. We will test the prevailing model that the 5'UTR adopts a distinct conformation prior to dimerization and packing. We will generate a more complete understanding of how Gag modulates the structure of the 5'UTR, thereby regulating viral replication.

摘要 HIV-1基因组的高度保守的5'非翻译区（UTR）在调节病毒免疫应答中起着重要作用。 复制的突破性的核磁共振实验，沿着重要的生化数据支持一个模型， 其中5 'UTR可以在至少两种构象状态之间转换：在一种状态下，基因组保持为构象状态， 单体，导致病毒基因的翻译;第二种状态是二聚体， 包装成组装的病毒体。因此，根据该模型，5 'UTR的构象 决定了每个基因组分子的命运病毒如何保持基因组的适当平衡 包装和翻译的命运仍然未知。但有重要证据表明，病毒蛋白， 以及潜在的宿主因素，促进5 'UTR的这些功能状态之间的切换。病毒 Gag蛋白特别地含有两个RNA结合结构域，核衣壳（NC）和基质（MA）结构域。 通过特异性相互作用，二聚化基因组被有效地募集到组装的病毒体中 在Gag的NC结构域和5 'UTR之间。作为核酸分子伴侣，NC促进蛋白质的折叠。 基因组转化为可能有利于二聚化和包装的有利构象。在 相反，MA结构域通过与NC的相互作用或通过调节 5 'UTR的结构。我们已经建立了一个单分子福斯特共振能量转移（smFRET） 使用成像方法来可视化5 'UTR的构象动力学。在我们的方法中， 携带供体和受体荧光团的分子被表面固定， 反射荧光（TIRF）显微镜。在这里，我们将进一步发展这种方法来阐明顺序 和5 'UTR构象变化、二聚化和Gag结合事件的时机。我们将测试流行的 模型表明，5 'UTR在二聚化和包装之前采用不同的构象。我们将产生一个更 完全理解Gag如何调节5 'UTR的结构，从而调节病毒复制。