Project 5: Structural investigation of lentiviral DNA integration

P5。摘要 逆转录病毒复制依赖于逆转录病毒DNA整合到宿主细胞染色体中。这 这一过程是由整合酶（IN）在一个稳定的突触复合体（称为整合体）的背景下催化的。的 整合体是IN链转移抑制剂（INSTI）的靶点，INSTI是目前唯一一类HIV IN拮抗剂 被批准用于治疗艾滋病近年来，人们对来自几种逆转录病毒物种的整合体进行了表征， 共同阐明了保守的负责整合的胞内核心组装体（CIC）。我们现在已经 建立了第一个基于Maedi-visna病毒（MVV）的慢病毒整合体模型， IN中不存在增溶或超活化突变的慢病毒整合的结构研究。的 在Cherepanov实验室通过单颗粒冷冻电镜对MVV整合体进行的初步研究表明， 比其非慢病毒对应物大得多，包含IN的十六聚体（四聚体的四聚体）。在 在这个项目中，我们利用新的系统来研究慢病毒DNA整合到染色质中 以及宿主细胞因子LEDGF/p75在此过程中的作用。作为该项目的一个翻译组成部分，我们 将开发一种慢病毒整合体模型，用于INSTI的结构研究和耐药机制。

P5. Abstract Retroviral replication depends on integration of reverse transcribed viral DNA into a host cell chromosome. This process is catalyzed by integrase (IN) in the context of a stable synaptic complex, known as the intasome. The intasome is the target for IN strand transfer inhibitors (INSTIs), the only class of HIV IN antagonists currently approved to treat HIV/AIDS. Recent years saw characterization of the intasomes from several retroviral species, collectively elucidating the conserved intasomal core assembly (CIC) responsible for integration. We have now established the first lentiviral intasome model based on maedi-visna virus (MVV), which affords detailed structural studies of lentiviral integration in the absence of solubilizing or hyperactivating mutations in IN. The preliminary study of the MVV intasome by single particle cryo-EM in the Cherepanov laboratory revealed that it is much larger than its non-lentiviral counterparts, comprising a hexadecamer (tetramer-of-tetramers) of IN. In the proposed project we take advantage of the new system to study lentiviral DNA integration into chromatin and the role of the host cell factor LEDGF/p75 in this process. As a translational component of this project, we will develop a lentiviral intasome model for structural studies of INSTIs and the mechanisms of drug resistance.