HIV nucleocapsid protein nucleic acid chaperone activity

描述（由申请人提供）：这项工作的总体目标是全面了解HIV核衣壳（NC）蛋白和Gag多蛋白前体的核酸（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，从而导致最热动力学稳定的结构。由于NC在病毒生命周期的几乎每个阶段都起作用，这项工作的影响很大。NC的NA结合和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开放性问题。在之前的资助期间，利用生化分析、集合和单分子生物物理方法，我们对HIV NC促进NA重排的机制有了新的认识。我们还在HIV Gag的背景下启动了NC的研究。我们发现Gag的伴侣活性需要NC结构域，令人惊讶的是，它是由肌醇磷酸（IP）结合到基质（MA）结构域刺激的。我们将继续采用创新的生物物理和生化方法来提高我们对NC伴侣活性机制的理解，并将扩大我们的研究，详细研究在Gag背景下相对较少的NC伴侣功能。我们对HIV MA调节Gag的伴侣特性的机制特别感兴趣。具体目的是：(1)探测HIV-1 NC WT、突变体和前体形式的NA伴侣活性；(2)探测HIV-1 Gag在体外和体内的伴侣活性。

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.