Role of HIV-1 IN during reverse transcription and uncoating

描述(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中的基本步骤。HIV-1IN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，人们对这种缺陷的潜在机制知之甚少。在体外，HIV-1IN与逆转录酶(RT)在物理上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，我们鉴定了IN上RT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测定了RT-IN复合体的亲和力和形成动力学。除了RT相互作用，我们最近还表征了HIV-1IN突变，这些突变导致核心产量低和不稳定，以及亲环素A(CypA)的掺入减少，CypA是一种特异性与衣壳(CA)结合的细胞肽-Pro异构酶。进一步的分析表明，通过维持CypA-CA的相互作用，在去包被过程中需要IN，这促进了病毒核心的最佳稳定性。综上所述，我们假设HIV-1IN可以通过与RT相互作用直接影响逆转录，也可以通过改变脱壳蛋白间接影响逆转录。这项应用的目的是为了更好地了解IN在去涂层和逆转录过程中的功能作用。具体目的是(1)确定HIV-1RT-IN相互作用的生物学意义，(2)表征HIV-1RT与IN之间的物理相互作用，并检测其他可调节RT-IN相互作用的因素，以及(3)了解IN影响病毒核心脱壳的机制。在目标1中，我们将通过破坏假定的IN-RT结合界面并评估其对反转录和病毒复制的影响，来测试感染过程中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IN突变的RT突变体，并使用基于荧光的高通量筛选来识别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止病毒复制。在目标2中，我们将使用靶向蛋白质足迹方法定位RT的IN相互作用结构域。我们还将研究与IN特异结合的宿主因子SIP1对RT-IN结合界面的影响，并确定SIP1是否可以影响IN在核进口和整合方面的活动。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梁因素。我们还将研究CypA-和TRIM51-与CA结合对IN在去包被过程中的作用的影响，以及去包被与逆转录的关系。在这个过程中，我们将阐明关键逆转录病毒蛋白和它们的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。相互作用的特征及其生物学意义的确定可能揭示IN的新功能作用，并确定新的抗HIV治疗的潜在靶点。

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.