HIV nucleocapsid protein nucleic acid chaperone activity

描述(由申请人提供)：这项工作的主要目标是全面了解HIV核衣壳(NC)蛋白和Gag多蛋白前体的核酸(NA)伴侣功能。NC的许多功能依赖于它的伴侣活性，即催化NA构象重排的能力，从而导致热力学上最稳定的结构。这项工作的影响很大，因为NC在病毒生命周期的几乎每个阶段都扮演着重要角色。NC的NA结合和伴侣功能已被证明在反转录、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在分子水平上理解许多这些过程的开放问题。在之前的赠款期间，我们使用生化分析以及集合和单分子生物物理方法，对HIV NC促进NA重排的机制获得了新的见解。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，并且令人惊讶的是，它被肌醇磷酸(IP)与基质(MA)结构域的结合所刺激。我们将继续使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范围，详细研究NC在GAG背景下相对较少了解的伴侣功能。我们对HIV MA调节GAG的伴侣特性的机制特别感兴趣。其具体目的是：(1)检测HIV-1Nc突变型、突变型和前体的NA伴侣活性；(2)检测HIV-1Gag的体内外伴侣活性。

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.