HIV nucleocapsid protein nucleic acid chaperone activity

描述（由申请人提供）：这项工作的首要目标是全面了解HIV核衣壳（NC）蛋白和Gag多聚蛋白前体的核酸（NA）伴侣功能。NC的许多功能依赖于其伴侣活性，即，催化NA构象重排的能力，导致最稳定的结构。由于NC在病毒生命周期的几乎每个阶段都发挥着重要作用，因此这项工作的影响力很大。NC的NA结合和分子伴侣功能已被证明在逆转录、整合、RNA包装和病毒组装中发挥重要作用，这些研究将解决我们对许多这些过程的分子水平理解中的未决问题。在上一个赠款期间，使用生化检测和合奏和单分子生物物理方法，我们获得了新的见解的机制，艾滋病毒NC促进NA重排。我们还在HIV Gag的背景下启动了NC的研究。我们发现，Gag的分子伴侣活性需要NC结构域，并且令人惊讶的是，通过磷酸肌醇（IP）结合到基质（MA）结构域来刺激。我们将继续采用创新的生物物理和生物化学方法，以提高我们对NC分子伴侣活性机制的理解，并将扩大我们的研究，详细调查相对知之甚少的NC分子伴侣功能的背景下Gag。我们特别感兴趣的是HIV MA调节Gag分子伴侣特性的机制。具体目标是：（1）探测HIV-1 NC的WT、突变体和前体形式的NA分子伴侣活性，和（2）探测HIV-1 Gag的体外和体内分子伴侣活性。

DESCRIPTION (provided by applicant): The overarching objective of this work is to gain a comprehensive understanding of the nucleic acid (NA) chaperone function of the HIV nucleocapsid (NC) protein and the Gag polyprotein precursor. Many of NC's functions rely on its chaperone activity, i.e., the ability to catalyze NA conformational rearrangements that lead to the most thermodynamically stable structure. The impact of this work is high due to NC's role in almost every stage of the viral lifecycle. NC's NA binding and chaperone function has been demonstrated to play an important role in reverse transcription, integration, RNA packaging, and viral assembly, and these studies will address open questions in our molecular level understanding of many of these processes. During the previous grant period, using biochemical assays and ensemble and single molecule biophysical approaches, we gained novel insights into the mechanism by which HIV NC facilitates NA rearrangements. We also initiated studies of NC in the context of HIV Gag. We discovered that Gag's chaperone activity requires the NC domain and surprisingly, is stimulated by inositol phosphate (IP) binding to the matrix (MA) domain. We will continue to employ innovative biophysical and biochemical approaches to improve our understanding of the mechanism of NC's chaperone activity, and will expand our studies to investigate in detail the relatively poorly understood chaperone function of NC in the context of Gag. We are particularly interested in the mechanism by which HIV MA modulates Gag's chaperone properties. The specific aims are: (1) To probe the NA chaperone activity of WT, mutant and precursor forms of HIV-1 NC, and (2) to probe HIV-1 Gag's chaperone activity in vitro and in vivo.