Visualization of HIV-1 integration in real time

HIV-1 整合的实时可视化

基本信息

批准号：
9425564
负责人：
KRISTINE E YODER
金额：
$ 4.23万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-12-15 至 2021-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9425564
关键词：
Address Affect Anti-Retroviral Agents Applications Grants Base Pairing Biochemical Biology Biophysical Process Chemistry Chromatin Chromosomes Complementary DNA Complex DNA DNA Structure DNA lesion Dimerization Drug Design Drug Targeting Energy Transfer Enzymes Event Exhibits Fluorescence Genome Goals HIV HIV-1 HIV-1 integrase Health Heterogeneity Histone H2A Histone H3 Histones Human Human T-Cell Leukemia Viruses Hydroxyl Radical Image Imagery In Vitro Infection Integrase Kinetics Label Lead Life Cycle Stages Link Long Terminal Repeats Lysine Magnetism Major Groove Mechanics Mediating Mutation Nucleoproteins Nucleosomes Nucleotides PWWP Domain Peptides Pharmaceutical Preparations Pharmacotherapy Physiological Post-Translational Protein Processing Process Protein Dynamics Proteins Reporting Research Resistance development Retroviridae Site Spumavirus Structure Technology Therapeutic Time Viral Virus Virus Integration analytical tool biophysical analysis cofactor comparative design dimer flexibility fluorophore imaging system in vivo innovation millisecond molecular imaging new therapeutic target novel pandemic disease prototype resistance mutation single molecule transcriptional coactivator p75 viral DNA wound

项目摘要

VISUALIZATION OF HIV-1 INTEGRATION IN REAL-TIME PROJECT SUMMARY / ABSTRACT Retroviral infections, including HIV and HTLV, continue to be a pandemic problem. While several drug therapies are able to treat HIV-1 infection, the virus's propensity to develop resistance mutations remains challenging. A clear priority for the HIV-1 treatment arsenal is to identifying novel drug targets. Stable integration of a retroviral donor cDNA into the host chromosome is absolutely required for a productive infection. Nearly three decades of research have established that retroviral integration is extremely inefficient in vivo and in vitro. Integration is catalyzed by the retrovirus encoded integrase (IN), which in many retroviruses forms a tetramer complex with the two viral cDNA long terminal repeat (LTR) ends (termed an intasome). The IN protein removes two 3' nucleotides and catalyzes end joining (strand transfer) of the resulting recessed 3' hydroxyls across one major groove of the target DNA separated by 4-6 bp. HIV-1 integration requires a host protein co-factor PSIP1/LEDGF/p75 for stable intasome assembly and to target integration to chromatin marked with the histone H3(K36) trimethylation post-translational modification (PTM). Remarkably, the prototype foamy virus (PFV) remains the only complete intasome structure. While anti- retroviral drugs that target the HIV-1 enzyme integrase also inhibit the PFV integrase, it remains unclear whether the integration mechanics of these respective retroviral intasomes are similar. We have used novel single molecule analytical tools to demonstrate that the PFV intasome catalyzes the two strand transfer events during integration in quick succession. We also visualized PFV intasomes on a linear target DNA and determined that the vast majority of IN-mediated search events were nonproductive. Together these observations suggested that target site selection limits PFV integration. We propose to extend our single molecule analysis to the biophysical mechanism of HIV-1 integration. Our preliminary studies have already identified significant differences between PFV and HIV-1, suggesting that the biophysical analysis of HIV-1 is likely to be more relevant to human health. Several important questions will be addressed in this new grant application: 1.) What are the protein dynamics of HIV-1 IN during the DNA target search and integration? 2.) What are the viral DNA dynamics during the DNA target search and integration process? 3.) What constitutes an efficient DNA target site? 4.) What factors influence the DNA target search process? 5.) What are the dynamics of intasome targeting on chromatin? We propose to utilize several innovative single molecule imaging systems to visualize HIV-1 integration in real- time. We will examine DNA lesions and mechanically altered DNA structures that may mimic the preferred target DNA configuration as well as chromatin containing histones with specific PTMs. These studies are designed to fully interrogate the animated processes associated with HIV-1 integration.

HIV-1 实时整合的可视化项目概要/摘要包括 HIV 和 HTLV 在内的逆转录病毒感染仍然是一个流行病。虽然有几种药物疗法能够治疗 HIV-1 感染，但该病毒产生耐药突变的倾向仍然存在具有挑战性的。 HIV-1 治疗方法的一个明确优先事项是确定新的药物靶点。逆转录病毒供体 cDNA 稳定整合到宿主染色体中对于生产来说是绝对必要的。感染。近三十年的研究表明逆转录病毒整合效率极低体内和体外。整合是由逆转录病毒编码的整合酶（IN）催化的，在许多情况下逆转录病毒与两个病毒 cDNA 长末端重复 (LTR) 末端（称为整合体）。 IN 蛋白去除两个 3' 核苷酸并催化末端连接（链转移）由此产生的 3' 羟基凹陷横跨目标 DNA 的一个主沟，间隔 4-6 bp。 HIV-1 整合需要宿主蛋白辅助因子 PSIP1/LEDGF/p75 来实现稳定的嵌体组装并靶向整合至带有组蛋白 H3(K36) 三甲基化翻译后修饰 (PTM) 标记的染色质。值得注意的是，原型泡沫病毒（PFV）仍然是唯一完整的嵌体结构。虽然反针对 HIV-1 酶整合酶的逆转录病毒药物也会抑制 PFV 整合酶，但目前尚不清楚这些各自的逆转录病毒嵌体的整合机制是否相似。我们使用新颖的单分子分析工具来证明 PFV 嵌体催化这两种整合过程中快速连续的链转移事件。我们还在线性上可视化了 PFV 整合体目标 DNA 并确定绝大多数 IN 介导的搜索事件都是非生产性的。一起这些观察结果表明，目标位置选择限制了 PFV 整合。我们建议延长我们的单一 HIV-1整合的生物物理机制的分子分析。我们的初步研究已经确定了 PFV 和 HIV-1 之间的显着差异，表明 HIV-1 的生物物理分析是可能与人类健康更相关。这项新的资助申请将解决几个重要问题：1.) 蛋白质动力学是什么 DNA 靶点搜索和整合过程中 HIV-1 IN 的影响？ 2.) 病毒 DNA 动态变化是怎样的？ DNA靶标搜索和整合过程？ 3.) 有效的 DNA 靶位点由什么构成？ 4.) 哪些因素影响 DNA 目标搜索过程？ 5.) 整合体靶向染色质的动力学是什么？我们提议利用几种创新的单分子成像系统来实时可视化 HIV-1 整合时间。我们将检查 DNA 损伤和可能模仿首选的机械改变的 DNA 结构。目标 DNA 配置以及含有具有特定 PTM 的组蛋白的染色质。这些研究是旨在全面询问与 HIV-1 整合相关的动画过程。