Project 3. Integration

项目3--集成 逆转录病毒复制需要将逆转录病毒DNA整合到宿主细胞染色体中。这 在含有多聚体的稳定核蛋白复合体中，整合酶(IN)催化这一过程 在病毒DNA末端组装的IN被称为内切体。体外组装肠系膜的特性研究 从许多逆转录病毒物种中阐明了保守的以及属特有的特征。慢病毒肠化体 同源低聚物中的大分子，含有多达16个亚基。然而，这个有趣的东西只表示一个 整合前复合体(PIC)的一小部分，它组装并调节细胞内的 在病毒感染的过程中会变得僵硬。此外，目前尚不清楚HIV-1 PIC如何与其靶标结合， 染色质宿主细胞基因组DNA。这个项目的目的是表征 使用自上而下和自下而上互补办法的艾滋病毒/慢病毒综合机制。自上而下- 接下来，我们将利用几种新型亲和试剂来从细胞提取液中浓缩PIC，然后进行研究 使用先进的DNA足迹和显微技术半纯化的天然HIV-1 PICS。实验 使用HIV-1 PICS将揭示IN结合伙伴LEDGF/p75及其同源表观遗传学的功能 染色质整合中的修饰(H3K36me3)。对于自下而上的方法，我们将采取 Maedi-Visna病毒(MVV，一种绵羊慢病毒)的套体的优势，与HIV/SIV套体不同， 可作为单分散制剂在体外组装。这种0.6-丙二醛复合体将使我们能够表征 慢病毒内切酶与染色质高时相复合体的动态和结构 和空间分辨率。拟议的研究将阐明慢病毒dna整合的体系结构。 以及它如何与宿主因子和染色体相互作用，同时避免自杀性的自我整合。 鉴于IN抑制剂在世界范围内被用于治疗HIV-1携带者，我们的结果将提供 这些非常成功的药物的靶标生物机器的前所未有的细节，并可能 揭示攻击PIC的新方法，以进行治疗干预。

Project 3 – Integration Retroviral replication requires integration of reverse-transcribed viral DNA into a host cell chromosome. This process is catalyzed by integrase (IN) in the context of the stable nucleoprotein complex, containing a multimer of IN assembled on viral DNA ends and termed the intasome. Characterization of in vitro-assembled intasomes from many retroviral species elucidated conserved as well as genus-specific features. Lentiviral intasomes harbor large IN homo-oligomers, containing as many as 16 subunits. However, the intasome represents only a small part of the pre-integration complex (PIC) that assembles and mediates intracellular trafficking of the intasome during virus infection. Furthermore, it is currently unclear how the HIV-1 PIC interfaces with its target, chromatinized host cell genomic DNA. This project aims to characterize the structure and properties of the HIV/lentiviral integration machinery using complementary top-down and bottom-up approaches. From the top- down, we will leverage several novel affinity reagents to enrich for PICs from cellular extracts and then study hemi-purified native HIV-1 PICs using advanced DNA footprinting and microscopy techniques. Experiments using HIV-1 PICs will reveal the function of the IN-binding partner LEDGF/p75 and its cognate epigenetic modification (H3K36me3) in integration in the context of chromatin. With the bottom-up approach, we will take advantage of the intasome from maedi-visna virus (MVV, an ovine lentivirus), which, unlike HIV/SIV intasomes, can be assembled in vitro as monodispersed preparations. This 0.6-MDa complex will allow us to characterize the dynamics and the structure of the complex between the lentiviral intasome and chromatin at high temporal and spatial resolutions. The proposed studies will shed light on the architecture of the lentiviral DNA integration apparatus and how it interfaces with host factors and chromosomes, while avoiding suicidal autointegration. Given that IN inhibitors are used worldwide to treat people living with HIV-1, our results will provide unprecedented details of the biological machine that is the target of these highly successful medicines and could reveal new ways to attack the PIC for therapeutic intervention.