Visualization of HIV-1 integration in real time

HIV-1整合的实时可视化 项目摘要/摘要 逆转录病毒感染，包括艾滋病毒和HTLV，仍然是一个大流行问题。虽然有几种药物 治疗方法能够治疗HIV-1感染，病毒产生抗药性突变的倾向仍然存在 很有挑战性。艾滋病毒-1治疗武器库的一个明显优先事项是确定新的药物靶点。 稳定地将逆转录病毒供体cdna整合到宿主染色体中是生产 感染。近三十年的研究已经证实，逆转录病毒整合是极其低效的 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 逆转录病毒与病毒的两个长末端重复序列形成一个四聚体复合体(称为AN 有趣的)。IN蛋白去除两个3‘核苷酸并催化末端连接(链转移) 得到的3‘羟基穿过靶DNA的一个主要凹槽，间隔4-6个碱基。HIV-1 整合需要宿主蛋白辅助因子PSIP1/LEDGF/p75以稳定地组装内切酶并靶向 整合到染色质，用组蛋白H3(K36)三甲基化翻译后修饰(PTM)标记。 值得注意的是，泡沫状病毒的原型(PFV)仍然是唯一完整的集合体结构。同时反- 针对HIV-1酶整合酶的逆转录病毒药物也抑制了PFV整合酶，目前尚不清楚 这些逆转录病毒内含体的整合机制是否相似。 我们已经使用新的单分子分析工具来证明pfv插入体催化这两个 整合过程中的链转移事件快速连续发生。我们还将PFV内含体视觉化于线性 以DNA为目标，并确定绝大多数IN中介的搜索事件都是非生产性的。同舟共济 这些观察结果表明，靶点选择限制了PFV的整合。我们建议延长我们的单人间 HIV-1整合生物物理机制的分子分析。我们的初步研究已经 发现了PFV和HIV-1之间的显著差异，这表明HIV-1的生物物理分析是 可能与人类健康更相关。 在这份新的赠款申请中，将解决几个重要的问题：1)什么是蛋白质动力学？ 在DNA靶标搜索和整合过程中HIV-1 IN？)病毒DNA的动态变化是什么 DNA目标搜索和整合过程？3)什么构成了有效的DNA靶点？有哪些因素？ 影响DNA目标搜索过程？)染色质上的端粒靶向动力学是什么？我们 建议利用几种创新的单分子成像系统来实时可视化HIV-1整合 时间到了。我们将检查dna损伤和机械改变的dna结构，它们可能会模仿首选的 靶DNA构型以及含有特定PTM的组蛋白的染色质。这些研究是 旨在全面审问与HIV-1整合相关的动画过程。

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.