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副本插入人类染色体。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一个不可逆步骤，细胞成为原病毒的永久载体（即病毒基因组的整合副本）。这个特性导致了病毒？持续和后来发展为艾滋病。然而，它也使HIV和其他逆转录病毒成为永久修复遗传疾病的天然基因载体。不幸的是，不受控制的改良逆转录病毒或载体整合可能导致重要基因的破坏或失调，从而导致癌症。因此，在LMO2基因附近插入逆转录病毒载体被认为是导致白血病的原因，11名参与X- scid (X染色体相关的严重联合免疫缺陷，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是由病毒编码的整合酶精心安排的，但许多细胞因子被病毒招募来帮助细胞内运输、保护并最终将病毒整合机制归巢到宿主细胞染色体DNA的合适区域。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，并能将其活性提高150倍。在细胞中，LEDGF可以保护HIV整合酶免受降解，并将病毒蛋白拴在染色体DNA上。所有可用的数据都有力地表明，LEDGF直接参与靶向HIV整合机制到活跃表达基因中。申请人最近进行的生化和结构研究解释了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特定逆转录病毒亚群（慢病毒科）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。这项研究将进一步加深我们对LEDGF在逆转录病毒复制中所起作用的理解。我们还将尝试定制LEDGF蛋白，将HIV整合机制重新定位到人类基因组的预定位置，这是创造更安全的基因治疗载体的一步。

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.