Visualization of HIV-1 integration in real time

HIV-1 整合的实时可视化

• 批准号: 10062830
• 负责人: KRISTINE E YODER
• 金额: $ 30.81万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-15 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10062830
• 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; Drug Design; Drug Targeting; Enzymes; Event; Exhibits; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; HIV; HIV Genome; HIV-1; HIV-1 integrase; Health; Heterogeneity; Histone H2A; Histone H3; Histones; Human; Human T-Cell Leukemia Viruses; Hydroxyl Radical; Image; 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; Visualization; 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

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整合的实时可视化 项目总结/摘要 逆转录病毒感染，包括艾滋病毒和HTLV，仍然是一个流行病问题。虽然几种药物 尽管目前的治疗方法能够治疗HIV-1感染，但病毒产生耐药性突变的倾向仍然存在， 挑战性HIV-1治疗武器库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是生产性逆转录病毒的绝对必要条件。 感染近三十年的研究已经证实，逆转录病毒的整合效率极低 在体内和体外。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， 逆转录病毒形成具有两个病毒cDNA长末端重复（LTR）末端的四聚体复合物（称为 intasome）。IN蛋白去除两个3'核苷酸并催化DNA的末端连接（链转移）。 产生跨越靶DNA的一个大沟的凹陷的3 ′羟基，其间隔4-6 bp。HIV-1 整合需要宿主蛋白辅因子PSIP 1/LEDGF/p75用于稳定的整合体组装和靶向 整合到染色质，标记有组蛋白H3（K36）三甲基化翻译后修饰（PTM）。 值得注意的是，原型泡沫病毒（PFV）仍然是唯一完整的整合体结构。虽然反- 靶向HIV-1酶整合酶的逆转录病毒药物也抑制PFV整合酶，目前尚不清楚 这些逆转录病毒整合体的整合机制是否相似。 我们已经使用了新的单分子分析工具来证明PFV整合体催化这两个过程， 在整合过程中快速连续的链转移事件。我们还在线性荧光显微镜上观察了PFV整合体。 靶DNA，并确定绝大多数IN介导的搜索事件是非生产性的。一起 这些观察结果表明靶位点的选择限制了PFV的整合。我们建议延长我们的单身 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 发现PFV和HIV-1之间存在显著差异，这表明HIV-1的生物物理分析是 可能与人类健康更相关。 在这个新的补助金申请中将解决几个重要问题：1。什么是蛋白质动力学 HIV-1在DNA靶点搜索和整合过程中的作用？2.）的情况。病毒DNA的动力学是什么 DNA目标搜索和整合过程？3.）第三章什么是有效的DNA靶位点？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 建议利用几种创新的单分子成像系统来可视化HIV-1在真实的中的整合， 时间我们将检查DNA损伤和机械改变的DNA结构，这些结构可能模仿首选的 靶向DNA构型以及含有组蛋白的染色质与特异性PTM。这些研究 旨在充分询问与HIV-1整合相关的动态过程。