Role of a Host Protein in HIV Particle Formation

描述(申请人提供)：艾滋病毒的早期工作侧重于了解这种病毒特有的蛋白质，并导致了针对这些病毒蛋白质的抗逆转录病毒药物的发现。近年来，研究转向了解在艾滋病毒生命周期的若干阶段起作用的宿主因素。对这些细胞蛋白的进一步了解可能会导致开发一类针对这种病毒与宿主相互作用的新型抗逆转录病毒药物。病毒生命周期中尚未确定病毒与宿主相互作用的一个重要阶段是病毒衣壳组装。我们最近发现了一种核苷酸结合宿主蛋白(HP68)，它似乎对未成熟衣壳形成中的翻译后事件至关重要。HP68与在包括HIV-I感染细胞在内的各种系统中组装HIV Gag链有关。当HP68中的截断突变体在哺乳动物细胞中表达时，HIV颗粒的形成通过显性的负机制显著减少。此外，无细胞组装系统中HP68的免疫耗竭会导致衣壳组装的翻译后阻断，而用纯化的HP68重建可以恢复完整衣壳的形成。综上所述，这些数据表明HP68在组装过程中发挥着重要作用。然而，要了解这种宿主因素在发病机制中的作用，还需要回答许多额外的问题。此前，HP68被认为是一种核糖核酸酶L抑制剂，可被艾滋病毒-1上调并阻断病毒核糖核酸的降解。我们的数据表明，HP68还有第二个功能，它还可以在翻译后促进衣壳的组装。为了验证这一假设，我们将首先使用定量分析来确定HP68构建体对病毒RNA水平、GAG合成和1124释放的影响。其次，将进行基础生化研究，以确定HP68是否与ATP或GTP结合，与膜结合，并与RNA结合。突变将被用来定位HP68中的结构域，以及参与HP68-GAG相互作用和衣壳形成的GAG结构域。通过这些研究，我们将能够确定ATP在HP68功能中的作用。最后，我们将研究HP68是否通过促进衣壳组装中间体的构象变化而发挥作用。将利用有关HP68与HIV-1 Gag结合的信息来试验阻断衣壳形成的方法。鉴于寻找新的抗逆转录病毒靶点和扩大高效抗逆转录病毒疗法(HAART)所取得的成果的重要性，IIP68是病毒产生的辅助因素的可能性是令人兴奋的。本文提出的研究将明确这种新的交易因子在HIV疾病中的作用，并为将来用于抗逆转录病毒药物筛选的检测奠定基础。

DESCRIPTION (provided by applicant): Early work on HIV focused on understanding proteins unique to this virus and led to the discovery of anti- retroviral agents that target these viral proteins. Recent years have seen a shift in research towards understanding host factors that act at a number of stages in the HIV life cycle. Further understanding of these cellular proteins could lead to the development of a new class of anti-retroviral agents that target such viral-host interactions. One important stage of the viral life cycle in which viral-host interactions have not yet been identified is viral capsid assembly. We have recently identified a nucleotide-binding host protein (HP68) that appears to be essential for post-translational events in immature capsid formation. HP68 is associated with assembling HIV Gag chains in a variety of systems, including HIV-I-infected cells. When a truncation mutant in HP68 is expressed in mammalian cells, HIV particle formation is dramatically reduced through a dominant negative mechanism. Furthermore, immunodepletion of HP68 from a cell-free assembly system results in post-translational blockade of capsid assembly, and recoristitution with purified HP68 restores formation of completed capsids. Together, these data indicate that HP68 plays an important role in assembly. However, to understand the role of this host factor for pathogenesis, many additional questions need to be answered. Previously, HP68 was identified as an RNase L inhibitor that is upregulated by HIV-1 and blocks viral RNA degradation. Our data indicate that HP68 has a second function in which it also acts post translationally to promote capsid assembly. 'To test this hypothesis, we will first define the effect of HP68 constructs on viral RNA levels, Gag synthesis, and 1124 release using quantitative assays. Secondly, basic biochemical studies will be performed to identify whether HP68 binds ATP or GTP, associates with membranes, and binds to RNA. Mutagenesis will be used to map domains in HP68 as well as domains in Gag that are involved in the HP68-Gag interaction and capsid formation. From these studies we will be able to determine the role of ATP in HP68 function. Finally, we will study whether HP68 acts by promoting a conformational change in capsid assembly intermediates. Information (concerning the binding of HP68 to HIV-1 Gag will be utilized to pilot methods for blocking capsid formation. The possibility that IiP68 is a co-factor for virus production is an exciting one, given the importance of finding new anti-retroviral targets and extending the gains obtained with highly active anti-retroviral therapy (HAART). The studies proposed here will define the role of this novel transacting factor in HIV disease, and lay the foundation for assays that could be used for anti-retroviral drug screening in the future.