Evolving understanding of HIV-1 reverse transcriptase structure, function, and inhibition.

在这笔赠款的前30年里，使用X射线结晶学，我们已经能够描述详细的 HIV-1逆转录酶(RT)在dsDNA、RNA/DNA和dsRNA复合体中的分子结构。 这些包括准备核苷酸掺入的结构、催化复合体和掺入后的结构。 移位前的结构。它们都代表了理解综合性质的关键状态 HIV感染过程中逆转录酶的表达，以及核苷RT的作用和耐药机制 抑制剂(NRTI)药物。在合作的努力下，我们的晶体结构使我们发现了两个 非核苷RT(NNRTI)药物(利培韦林和依特拉韦林)，导致五种获得许可的抗艾滋病药物。 在之前的资助期间，主要的亮点包括用dsRNA解决复合体中的RT结构 核苷酸掺入前的起始复合体，与NNRTI的RT/DNA，与NRTI药物的RT，以及与 核苷酸竞争逆转录酶抑制剂(NcRTI)，包括INDOPY-1。我们建议通过确定一个 一系列RT/DNA-RNA/RNA结构，以绘制第一链DNA合成如何启动的分子细节， 重点关注伴随着从启动过渡而来的RT和核酸的构象变化 (慢的)到(快的)DNA合成。同样，第二链起始的细节将从一开始就进行探讨 具有与DNA模板对面的多嘌呤-链RNA引物的RT结构。额外的HIV-1 RT 将确定含有药物和研究用抑制剂的结构，以进一步了解其作用机制 抑制和耐药性，以及可能出现更多目前不是抗艾滋病药物靶点的部位。 虽然HIV蛋白和酶最初是作为Gag-Pol前体多蛋白的一部分产生的， 对于成熟前的不成熟形式的详细结构和功能，人们知之甚少。 最近，我们已经生产了多毫克量的HIV-1 Gag-Pol和Pol前体多蛋白 这是第一次。冷冻-EM检测HIV-1 POL多聚体蛋白显示RT为二聚体结构 其核心为p66/p51异二聚体构型，表明成熟RT结构的形成是 从其形态发生的早期阶段开始。RT二聚体核心的形成也带来了蛋白酶 将单体转化为二聚体排列，从而为RT在蛋白酶中的潜在作用提供了结构洞察力 病毒粒子成熟时的激活。我们将进一步进行低温EM、结晶学和生物物理研究 HIV-1 POL多聚蛋白及其与相关结合伙伴的复合体提供进一步的结构和 关于病毒组装和成熟的各个方面的生物物理见解。 拟议的工作依赖于与Jeffrey DeStefano(马里兰州)和Stephen Hughes(NCI- 弗雷德里克)，Mamuka Kvaratskhelia(科罗拉多-丹佛)，Ronald Levy(坦普尔)，Dmitry Lyumkis(Salk)，Stefan 萨拉菲亚诺斯(埃默里)和吉尔达·塔切德健(莫纳什)。

During the first 30 years of this grant, using X-ray crystallography, we have been able to describe the detailed molecular architecture of HIV-1 reverse transcriptase (RT) in complex with dsDNA, RNA/DNA, and dsRNA. These include structures poised for nucleotide incorporation, catalytic complexes, and post-incorporation structures prior to translocation. All of them represent key states for understanding the comprehensive nature of reverse transcription during HIV infection, and the mechanisms of action of and resistance to nucleoside RT inhibitor (NRTI) drugs. In a collaborative effort, our crystallographic structures enabled the discovery of two non-nucleoside RT (NNRTI) drugs (rilpivirine and etravirine), leading to five licensed anti-AIDS medications. During the previous grant period, major highlights included solving RT structures in complex with a dsRNA initiation complex prior to nucleotide incorporation, RT/DNA with an NNRTI, RT with NRTI drugs, and RT with nucleotide-competing RT inhibitors (NcRTIs) including INDOPY-1. We propose to follow up by determining a series of RT/DNA-RNA/RNA structures to map molecular details of how first-strand DNA synthesis is initiated, with a focus on conformational changes in RT and nucleic acid that accompany the transition from initiation (slow) to processive (fast) DNA synthesis. Similarly, details of second-strand initiation will be probed beginning with a structure of RT with the polypurine-tract RNA primer across from a DNA template. Additional HIV-1 RT structures with drugs and investigational inhibitors will be determined to further understand mechanisms of inhibition and resistance, and the potential for additional sites not currently targeted by anti-AIDS drugs. Although the HIV proteins and enzymes are produced initially as part of Gag-Pol precursor polyproteins, relatively little is known about the detailed structure and function of the immature forms prior to maturation. Recently, we have produced multiple-milligram amounts of the HIV-1 Gag-Pol and Pol precursor polyproteins for the first time. Cryo-EM determination of the HIV-1 Pol polyprotein shows a dimeric structure with RT in a p66/p51 heterodimer-like configuration at its core, suggesting that formation of the mature RT structure is guided from early stages of its morphogenesis. Formation of the RT dimeric core also brings the protease monomers into a dimeric arrangement, thus providing structural insight into the potential role of RT in protease activation during virion maturation. We will pursue further cryo-EM, crystallographic, and biophysical studies of the HIV-1 Pol polyprotein and its complexes with relevant binding partners to provide further structural and biophysical insights that shed light on various aspects of virus assembly and maturation. The proposed work relies on collaborations with Jeffrey DeStefano (Maryland), and Stephen Hughes (NCI- Frederick), Mamuka Kvaratskhelia (Colorado-Denver), Ronald Levy (Temple), Dmitry Lyumkis (Salk), Stefan Sarafianos (Emory), and Gilda Tachedjian (Monash).