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

对 HIV-1 逆转录酶结构、功能和抑制的理解不断发展。

• 批准号: 9927362
• 负责人: EDWARD ARNOLD
• 金额: $ 71万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-11-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9927362
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Anti-HIV Agents; Antiviral Agents; Architecture; Bacteria; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biophysics; Chemical Structure; Clinical; Collaborations; Colorado; Combined Modality Therapy; Complement; Complex; Computer Models; Consensus; Cryoelectron Microscopy; Crystallization; Crystallography; DBL Oncoprotein; DNA; DNA biosynthesis; Data; Deuterium; Double-Stranded RNA; Drug Design; Drug Targeting; Drug resistance; Engineering; Enzymes; Grant; HIV; HIV Infections; HIV-1; Highly Active Antiretroviral Therapy; Hydrogen; Investigation; Knowledge; Label; Letters; Light; Maps; Maryland; Metals; Methodology; Methods; Molecular; Molecular Biology; Molecular Conformation; Molecular Probes; Morphogenesis; Nature; Nucleic Acids; Nucleosides; Nucleotides; Peptide Hydrolases; Pharmaceutical Preparations; Polyproteins; Production; Protein Engineering; Proteins; Publications; RNA; RNA primers; RNA-Directed DNA Polymerase; Research; Research Personnel; Resistance; Reverse Transcriptase Inhibitors; Reverse Transcription; Role; Sampling; Series; Site; Structure; System; Time; Treatment Protocols; Viral; Virion; Virus Assembly; Work; X-Ray Crystallography; biophysical analysis; biophysical properties; biophysical techniques; data submission; design; design and construction; dimer; drug action; ds-DNA; experimental study; follow-up; improved; inhibitor/antagonist; innovation; insight; light scattering; milligram; monomer; non-nucleoside reverse transcriptase inhibitors; novel strategies; nucleic acid structure; nucleoside inhibitor; pol Gene Products; pre-exposure prophylaxis; protein expression; resistance mutation; small molecule

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).

在这项资助的前30年里，利用X射线晶体学，我们已经能够详细描述 HIV-1逆转录酶（RT）与dsDNA、RNA/DNA和dsRNA复合的分子结构。 这些包括准备用于核苷酸掺入、催化复合物和掺入后的结构。 在易位之前的结构。所有这些都代表了理解全面性质的关键状态 逆转录病毒感染过程中，和核苷RT的作用和耐药机制 抑制剂（NRTI）药物。在一次合作努力中，我们的晶体结构使我们能够发现两个 非核苷RT（NNRTI）药物（利匹韦林和依曲韦林），导致五种获得许可的抗艾滋病药物。 在上一个资助期间，主要的亮点包括解决与dsRNA复合的RT结构 核苷酸掺入前的起始复合物，用NNRTI的RT/DNA，用NRTI药物的RT，和 核苷酸竞争性RT抑制剂（NcRTI），包括INDOPY-1。我们建议采取后续行动， 一系列RT/DNA-RNA/RNA结构，用于绘制第一链DNA合成如何启动的分子细节， 重点关注RT和核酸的构象变化，这些变化伴随着从起始 （慢）到进行性（快）DNA合成。类似地，第二链起始的细节将从 具有RT的结构，其中多嘌呤段RNA引物与DNA模板相对。其他HIV-1 RT 将确定药物和研究抑制剂的结构，以进一步了解 抑制和耐药性，以及目前抗艾滋病药物未靶向的其他位点的潜力。 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， 对于成熟前未成熟形式的详细结构和功能知之甚少。 最近，我们已经生产了数毫克量的HIV-1 Gag-Pol和Pol前体多蛋白 第一次HIV-1 Pol多聚蛋白的Cryo-EM测定显示二聚体结构， p66/p51异源二聚体样构型在其核心，表明成熟RT结构的形成是 从其形态发生的早期阶段开始引导。RT二聚体核心的形成也带来了蛋白酶 单体转化为二聚体的安排，从而提供结构洞察RT在蛋白酶的潜在作用， 在病毒体成熟期间激活。我们将继续进行进一步的冷冻电镜，晶体学和生物物理学研究， HIV-1 Pol多蛋白及其与相关结合配偶体的复合物，以提供进一步的结构和 生物物理的见解，揭示了病毒组装和成熟的各个方面。 拟议的工作依赖于与杰弗里·德斯特凡诺（马里兰州）和斯蒂芬·休斯（NCI- 弗雷德里克）、马穆卡·克瓦拉茨克赫利亚（科罗拉多-丹佛）、罗纳德·利维（坦普尔）、德米特里·卢姆基斯（索尔克）、斯特凡 Sarafianos（埃默里）和Gilda Tachedjian（莫纳什）。