Role of HIV-1 IN during reverse transcription and uncoating

描述（由申请人提供）：逆转录病毒整合酶（IN）催化将病毒基因组的双链DNA拷贝整合到受感染细胞的染色体上的关键步骤。HIV-1 IN的某些突变可以特异性地损害逆转录，而对生命周期的其他步骤没有明显影响。然而，这一缺陷的潜在机制尚不清楚。在体外，HIV-1 In与逆转录酶（RT）物理相互作用并刺激逆转录，但这种RT- In相互作用的生物学相关性尚不清楚。利用核磁共振波谱技术，我们确定了RT-IN相互作用的表面，并利用基于表面等离子体共振的生物传感器确定了RT-IN复合物形成的亲和力和动力学。除了RT相互作用外，我们最近还描述了HIV-1 In突变导致核心产量低和不稳定性，以及亲环蛋白A （CypA）的掺入减少，CypA是一种与衣壳特异性结合的细胞肽基脯氨酸异构酶（CA）。进一步的分析表明，IN在脱膜过程中需要维持CypA-CA的相互作用，从而促进病毒核心的最佳稳定性。综上所述，我们假设HIV-1 IN可以通过与RT相互作用直接影响逆转录，也可以通过改变脱膜间接影响逆转录。本应用程序的目的是为了更好地了解IN在脱膜和逆转录过程中的功能作用。具体目的是：(1)确定HIV-1 RT-IN相互作用的生物学意义，(2)表征HIV-1 RT和IN之间的物理相互作用，并检查可以调节RT-IN相互作用的其他因素，以及(3)了解IN影响病毒核心脱壳的机制。在Aim 1中，我们将通过破坏假定的In - rt结合界面和评估对逆转录和病毒复制的影响来测试感染期间RT-IN相互作用的生物学相关性。我们还将筛选能够补偿RT-非相互作用的IN突变的RT突变体，并使用基于荧光的高通量筛选来鉴定RT-IN相互作用的小分子抑制剂，并确定这些抑制剂是否也阻断病毒复制。在Aim 2中，我们将使用靶向蛋白足迹方法绘制RT的In相互作用域。我们还将研究宿主因子SIP1对RT-IN结合界面的影响，并确定SIP1是否会影响IN在核导入和整合中的活性。宿主因子SIP1是逆转录所必需的。在目标3中，我们将确定病毒核心中的In - ca相互作用是否需要翻译后修饰或桥接因子。我们还将研究CypA-和trim51与CA结合对IN在脱衣过程中的作用的影响，以及脱衣与逆转录之间的关系。在此过程中，我们将阐明关键逆转录病毒蛋白与其宿主因子之间的相互作用，以及这种相互作用对逆转录和脱壳的影响。表征相互作用并确定其生物学意义可能揭示IN的新功能作用，并确定抗hiv治疗的新潜在靶点。

DESCRIPTION (provided by applicant): Retroviral integrase (IN) catalyzes the essential step of integrating the double-stranded DNA copy of the viral genome into the chromosome of an infected cell. Certain mutations of HIV-1 IN can specifically impair reverse transcription with no apparent effects on other steps in the life cycle. However, the underlying mechanism for this defect is poorly understood. In vitro, HIV-1 IN physically interacts with reverse transcriptase (RT) and stimulates reverse transcription, but the biological relevance of this RT-IN interaction is not known. Using nuclear magnetic resonance spectroscopy, we have identified the RT-interacting surface on IN, and have determined the affinity and kinetics of RT-IN complex formation by using a surface plasmon resonance-based biosensor. In addition to RT interaction, we recently have characterized HIV-1 IN mutations that resulted in poor core yield and instability, and reduced incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to capsid (CA). Further analyses indicate that IN is required during uncoating by maintaining CypA-CA interaction, which promotes optimal stability of the viral core. Taken together, we hypothesize that HIV-1 IN can affect reverse transcription either directly by interacting with RT or indirectly by altering uncoating. The goal of this application is to gain a better understanding of the functional role of IN during uncoating and reverse transcription. The specific aims are (1) to determine the biological significance of the HIV-1 RT-IN interaction, (2) to characterize the physical interaction between HIV-1 RT and IN, and examine other factors that can modulate the RT-IN interaction, and (3) to understand the mechanism by which IN affects the uncoating of viral cores. In Aim 1, we will test the biological relevance of the RT-IN interaction during infection by disrupting the putative IN-RT binding interface and assessing the impact on reverse transcription and viral replication. We will also screen for RT mutants that can compensate for the RT- noninteracting IN mutations, and use a fluorescence-based high-throughput screen to identify small-molecule inhibitors of the RT-IN interaction and determine whether such inhibitors also block viral replication. In Aim 2, we will map the IN-interacting domain of RT using a targeted protein footprinting method. We will also examine the effect of a host factor SIP1, which binds specifically to IN and is required for reverse transcription, on the RT-IN binding interface, and determine if SIP1 can affect IN's activities on nuclear import and integration. In Aim 3, we will determine if the IN-CA interaction in the viral core requires post-translational modification or a bridge factor. We will also examine the effect of CypA- and TRIM51-binding to CA on IN's role during uncoating, and the relationship between uncoating and reverse transcription. In the process, we will shed light on the interactions among key retroviral proteins and their host factors, and the effect of such interactions on reverse transcription and uncoating. Characterization of the interactions and determination of their biological significance may reveal new functional roles for IN, and identify new potential targets for anti-HIV therapy.