Structural dynamics of the HIV-1 genomic 5' UTR

摘要 HIV-1基因组高度保守的5‘非翻译区在调节病毒的过程中起着核心作用 复制。开创性的核磁共振实验以及重要的生化数据支持 其中5‘非编码区可以在至少两种构象状态之间转换：在一种状态下，基因组保持一种 单体，导致病毒基因的翻译；第二状态适合二聚化和 包装成组装的病毒粒子。因此，根据这一模型，5‘非编码区的构象 决定着每个基因组分子的命运。病毒如何维持适当的基因组平衡 包装和翻译的命运仍不得而知。但有重要证据表明，病毒蛋白，如 以及潜在的宿主因素，促进了5‘非编码区的这些功能状态之间的切换。病毒式传播 特别是GAG蛋白含有两个RNA结合域，即核衣壳(NC)和基质(MA)结构域。 通过特定的相互作用，将二聚化的基因组有效地招募到组装的病毒粒子中 在GAG的NC结构域和5‘非编码区之间。作为一种核酸伴侣，NC促进了DNA的折叠 基因组转化为热力学上有利的构象，可能有利于二聚化和包装。在……里面 相比之下，MA结构域通过与NC的相互作用或通过调节 5‘非编码区的结构。我们建立了单分子Förster共振能量转移(SmFRET) 成像方法可视化5‘非编码区的构象动力学在我们的方法中，单个UTR 携带供体和受体荧光团的分子被表面固定并与全内 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 以及5‘UTR构象变化、二聚化和Gag结合事件的时间。我们将测试盛行的 5‘非编码区在二聚化和堆积之前采用不同构象的模型。我们将产生更多 完全了解GAG是如何调节5‘非编码区的结构，从而调控病毒复制的。

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.