HIV GAG PRECURSOR PROTEIN INTERACTIONS

描述（改编自申请人的摘要）： 这些研究是为了表征HIV颗粒的结构。一般来说，HIV 颗粒采用两种不同的形态：未成熟的和蛋白水解的 处理成熟的形式。未成熟和成熟HIV的自然变异性 病毒粒子使得研究这些粒子形式变得极其困难。然而，在这方面， 很明显，HIV Gag蛋白构成了 这两种病毒形式的决定因素。具体地，Gag前体（PrGag） 蛋白质指导未成熟病毒体在细胞膜上的组装， 加工的Gag蛋白基质（MA）、衣壳（CA）、p2、核衣壳（NC）、p1 和p6组织成熟的病毒装配体。为了了解HIV Gag蛋白 为了形成更高级的结构，我们最近开发了用于 组装在脂质膜上的Gag蛋白的体外分析。 值得注意的是，我们的结果与体内观察结果一致， 提供了关于PrGag组装如何发生的新见解。调查 本提案中所描述的旨在扩展我们有前途的初步 结果，并测试我们的预测使用病毒颗粒产生的， 哺乳动物细胞我们获得的结果将提供重要的见解， 组装和形态发生的机制，应该作为基础， 攻击HIV病毒的核心我们的具体目标如下：1.电子 Gag蛋白在脂质膜上组装的衍射图像分析：我们 将采用我们的新方法组装组氨酸标记的艾滋病毒 Gag蛋白在含有镍螯合脂质的膜上，以获得 膜结合Gag蛋白的三维结构及其 交互. 2.体内结构模型的验证： 从体外研究中获得的结果将使用细胞来源的 病毒颗粒，将进行生物病理学、生物化学和 从遗传学上评估结构模型的准确性和有用性。

DESCRIPTION (Adapted from applicant's abstract): The ultimate objective of these investigations is to characterize HIV particle structures. Typically, HIV particles adopt two distinct morphologies: immature, and proteolytically processed mature forms. The natural variability of immature and mature HIV virions has made it extremely difficult to study these particle forms. However, it is clear that the HIV Gag proteins constitute the major structural determinants of both viral forms. Specifically, the Gag precursor (PrGag) proteins direct the assembly of immature virions on cellular membranes, while the processed Gag proteins matrix (MA), capsid (CA), p2, nucleocapsid (NC), p1, and p6 organize mature virus assemblies. To understand how HIV Gag proteins associate to form higher order structures, we recently developed methods for the in vitro analysis of Gag proteins assembled on lipid membranes. Significantly, our results are consistent with in vivo observations, and have provided new insights as to how PrGag assembly occurs. The investigations described in this proposal are designed to extend our promising preliminary results, and to test our predictions using virus particles produced from mammalian cells. The results we obtain will provide crucial insights into mechanisms of assembly and morphogenesis that should serve as a foundation to attack HIV at its viral core. Our specific aims are as follows: 1. Electron diffraction-image analysis of Gag proteins assembled on lipid membranes: We will employ our novel method of assembling histidine-tagged (his-tagged) HIV Gag proteins on membranes containing nickel-chelating lipids to obtain three-dimensional structures of membrane-bound Gag proteins and their interactions. 2. Validation of structural models in vivo: The validity of results obtained from in vitro studies will be tested using cellularly derived virus particles, which will be examined biophysically, biochemically, and genetically to assess the accuracy and usefulness of structural models.