LEDGF/p75 in retroviral DNA integration: from structure to function to potential applications

与其他逆转录病毒类似，艾滋病毒必须将其基因组的DNA复制品插入人类染色体中。逆转录病毒DNA整合定义了病毒和受感染细胞生命周期中的不可逆步骤，细胞成为前病毒的永久载体（即病毒基因组的整合拷贝）。这一特点有助于病毒？持续存在，后来发展为艾滋病。然而，它也使艾滋病毒和其他逆转录病毒成为永久修复遗传疾病的天然基因传递载体。不幸的是，不受控制的整合修改逆转录病毒或？带菌者可能导致重要基因的破坏或失调，并导致癌症。因此，在LMO 2基因附近插入逆转录病毒载体被认为是白血病的原因，这些白血病影响了参加X-SCID（X染色体连锁严重联合免疫缺陷，也称为？泡泡男孩病？）基因治疗试验 尽管与整合相关的基本酶促步骤由病毒编码的酶整合酶协调，但病毒募集许多细胞因子以帮助病毒整合机制的细胞内转运、保护和最终归巢到宿主细胞染色体DNA的合适区域上。LEDGF是一种人类染色体相关蛋白，广泛参与HIV和相关非灵长类病毒的整合。LEDGF以高亲和力结合整合酶蛋白，并且能够将其活性增加多达150倍。在细胞中，LEDGF可以保护HIV整合酶免受降解，并将病毒蛋白质与染色体DNA结合。所有可用的数据强烈表明，LEDGF直接参与靶向HIV整合机制进入活跃表达的基因。申请人最近进行的生物化学和结构研究解释了LEDGF-IN识别的一些关键方面。目前的研究项目旨在解释LEDGF介导的整合激活机制及其对逆转录病毒（慢病毒科）特定子集的选择性。为此，我们将尝试获得晶体，并确定各种整合酶-LEDGF复合物的分子结构。这项研究将进一步了解LEDGF在逆转录病毒复制中的作用。我们还将尝试定制LEDGF蛋白，以将HIV整合机制重新定位到人类基因组的预定义位点，这是朝着创建更安全的基因治疗载体迈出的一步。

Akin to other retroviruses, HIV must insert a DNA replica of its genome into a human chromosome. Retroviral DNA integration defines a step of no return in the life cycle of the virus and the infected cell, the cell becoming a permanent carrier of the provirus (i.e. integrated copy of the viral genome). This feature contributes to the virus? persistency and later progression to AIDS. However, it also makes HIV and other retroviruses natural gene delivery vehicles for permanent repair of genetic disorders. Unfortunately, uncontrolled integration of modified retroviruses or ?vectors? can result in disruption or untuning of important genes and lead to cancer. Thus, insertion of a retroviral vector in proximity of the LMO2 gene is thought to be the cause of leukemias that affected three out of eleven children that participated in an X-SCID (X chromosome-linked severe combined immunodeficiency, also known as ?Bubble Boy Disease?) gene therapy trial. Although the essential enzymatic steps associated with integration are orchestrated by virally-encoded enzyme integrase, many cellular factors are recruited by the virus to aid the intracellular transport, protection and finally homing of the viral integration machinery onto a suitable region of the host cell chromosomal DNA. LEDGF is a human chromosome-associated protein that has been extensively implicated in integration of HIV and related non-primate viruses. LEDGF binds integrase protein with high affinity and is capable of increasing its activity as much as 150-fold. In cells, LEDGF can protect HIV integrase from degradation and tether the viral protein to chromosomal DNA. All available data strongly indicate that LEDGF is directly involved in targeting HIV integration machinery into actively expressed genes. Recent biochemical and structural studies conducted by the applicant explained some key aspects of the LEDGF-IN recognition. The current research project is aimed to explain mechanism of LEDGF-mediated activation of integration and its selectivity towards a specific subset of retroviruses (lentiviridae). To this end we will try to obtain crystals and determine molecular structures of various integrase-LEDGF complexes. The study will further our understanding of the role that LEDGF plays in retroviral replication. We will also attempt to tailor LEDGF protein to re-target HIV integration machinery to a predefined spot of the human genome, a step towards creating safer gene therapy vectors.