Structural understanding of the HIV-1 reverse transcription initiation process

摘要 在逆转录病毒中，逆转录是从形成的分子间双链引物开始的。 通过核衣壳驱动的基因组U5-引物结合位点(U5-PBS)区域的退火 与宿主tRNA结合。然而，这个关键复合体的结构，以及逆转录酶是如何 (RT)与之相互作用在很大程度上是未知的。文中提出的HIV-1复合体的结构 提案显示，U5-PBS中的四个串联GNRA模块通过以下方式在空间上组织建筑群 进行连续的四环-受体对接相互作用：一个参与重排的tRNA，一个 隔离起始点，两个隔离18个碱基的引物以抑制RT结合。因此，在 与广泛接受的单个RT分子识别这种复合体的模型不同 启动转录，我们的数据表明，事实上，需要两个RT分子结合在一个 以循序渐进的方式释放被压抑的状态并确保准确的启动：第一个广泛 与U5茎相互作用并充当重建器，允许后续的茎绑定 标准的18个碱基的引物，并执行酶活性。对体系结构的操纵， 重塑过程，或与核衣壳的竞争，导致严重的起爆精度损失。 因此，这项研究重新定义了我们对艾滋病毒逆转录启动的基本理解；分配 RT是一种结构重构者角色，与其酶功能分开；并表明 机制可能有助于控制病毒粒子中DNA合成的开始。目标将是： (#1)通过突变分析进一步详细说明机制，(#2)了解结构作用 以确定与NC结合的重构体RT和酶RT的结构 U5-PBS：tRNAlys复合体。

Abstract In retroviruses, reverse transcription is initiated from an intermolecular duplex primer formed by nucleocapsid-driven annealing of the U5-primer binding site (U5-PBS) region of the genome with a host tRNA. However, the structure of this critical complex, and how reverse transcriptase (RT) interacts with it is largely unknown. The structure of the HIV-1 complex presented in this proposal shows that four tandem GNRA modules in U5-PBS spatially organizes the complex by making continuous tetraloop-receptor docking interactions: one engages a rearranged tRNA, one sequesters the initiation site, and two sequester the 18-bp primer to inhibit RT binding. Thus, in contrast to the widely accepted model that a single RT molecule recognizes this complex to initiate transcription, our data show that, in fact, two molecules of RT are required to bind in a step-wise manner to release the repressed state and ensure accurate initiation: the first extensively interacts with the U5 stem and acts as a remodeler, allowing for the subsequent one to bind the canonical 18-bp primer and perform the enzymatic activity. Manipulation of the architecture, the remodeling process, or competition with nucleocapsid, leads to severe loss of initiation accuracy. Thus, this study redefines our basic understanding of HIV reverse transcription initiation; assigns RT a structural remodeler role, separate from its enzymatic function; and indicates that the unique mechanism may contribute to the control of start of DNA synthesis in virions. The aims will be: (#1) to further detail the mechanism by mutational analysis, (#2) to understand the structural role of NC and (#3) to determine the structures of the remodeler RT and enzymatic RT bound to the U5-PBS:tRNAlys complex.