Single Molecule HIV-1 NC/Gag-DNA Interactions

描述（由申请人提供）：这项工作的目的是研究HIV-1和其他逆转录病毒Gag和核衣壳蛋白（NC）在逆转录病毒系统中涉及核酸结构转换和病毒自组装过程的调节中的作用。PI率先开发了新的单分子核酸（NA）拉伸方法，可用于定量探测核酸结构重排和蛋白质-核酸相互作用。拟议的工作继续开发新的方法来测试特定的假设，核酸-蛋白质相互作用是重要的逆转录病毒包装和复制。这些研究的结果与体内协作工作相结合。NC重排核酸以促进逆转录的能力，称为NA分子伴侣活性，对于逆转录病毒复制是必不可少的。NA拉伸方法特别适合于探测NA分子伴侣活性，因为可以直接控制核酸结构重排和包装，并且可以量化所得响应。将开发一种新的方法来机械探测逆转录和表征对HIV-1复制重要的蛋白质对这一过程的影响。具体目标是：（1）定量野生型和突变型HIV-1和其他逆转录病毒核酸伴侣蛋白与单链和双链DNA和特定RNA结构的相互作用。这项工作将测试的假设，核酸分子伴侣活性是由三个关键要素：核酸聚集，核酸不稳定，和快速蛋白质-核酸相互作用动力学。(2)表征可能干扰逆转录的APOBEC蛋白的DNA相互作用。我们推测APOBEC蛋白通过改变特定的DNA相互作用来抑制逆转录。为了验证这一假设，将研究人类APOBEC与单链和双链DNA相互作用的热力学和动力学。(3)定量野生型和突变型HIV-1 Gag的核酸相互作用。虽然HIV-1 NC是Gag的结构域，主要负责其与NA的相互作用，但这些蛋白质促进细胞中显著不同的过程。这一目标旨在了解NC和Gag如何表现出不同的NA相互作用，以及这些相互作用如何改变逆转录和NA包装过程。(4)在NC和APOBEC 3G存在下机械测量逆转录酶（RT）聚合，并确定它们如何改变RT的基本过程。 公共卫生相关性：核酸包装和逆转录是逆转录病毒复制的关键组成部分。该项目开发了表征这些过程的新方法，以促进对逆转录病毒复制的分子水平理解的发展，这反过来将有助于开发针对这些过程的药物。

DESCRIPTION (provided by applicant): The objective of this work is to investigate the role of HIV-1 and other retroviral Gag and nucleocapsid proteins (NCs) in the regulation of processes involving nucleic acid structural transitions and viral self assembly in retroviral systems. The PI has pioneered the development of new single molecule nucleic acid (NA) stretching methods that can be used to quantitatively probe nucleic acid structural rearrangements and protein-nucleic acid interactions. The proposed work continues to develop novel methods to test specific hypotheses concerning nucleic acid-protein interactions that are important for retroviral packaging and replication. The results of these studies are integrated with collaborative in vivo work. The capability of NCs to rearrange nucleic acids to facilitate reverse transcription, referred to as NA chaperone acitivity, is essential for retroviral replication. NA stretching methods are uniquely well suited for probing NA chaperone activity because nucleic acid structural rearrangement and packaging can be directly controlled and the resulting response can be quantified. A new method will be developed to mechanically probe reverse transcription and characterize the effects of proteins important for HIV-1 replication on this process. The specific aims are: (1) To quantify the interactions of wild type and mutant HIV-1 and other retroviral nucleic acid chaperone proteins with single- and double-stranded DNA and specific RNA structures. This work will test the hypothesis that nucleic acid chaperone activity is comprised of three critical elements: nucleic acid aggregation, nucleic acid destabilization, and rapid protein-nucleic acid interactions kinetics. (2) To characterize the DNA interactions of APOBEC proteins that may interfere with reverse transcription. We hypothesize that APOBEC proteins inhibit reverse transcription by altering specific DNA interactions. To test this hypothesis, will examine the thermodynamics and kinetics of human APOBEC interactions with single- and double-stranded DNA. (3) To quantify the nucleic acid interactions of wild type and mutant HIV-1 Gag. Although HIV-1 NC is the domain of Gag that is primarily responsible for its interactions with NAs, these proteins facilitate significantly different processes in the cell. This aim seeks to understand how NC and Gag exhibit different NA interactions and how these interactions alter reverse transcription and the NA packaging process. (4) To mechanically measure reverse transcriptase (RT) polymerization in the presence of NC and APOBEC3G and determine how they alter RT's essential processes. PUBLIC HEALTH RELEVANCE: Nucleic acid packaging and reverse transcription are critical components of retroviral replication. This project develops novel methods for characterizing these processes to contribute towards development of a molecular level understanding of retroviral replication, which in turn will help to develop drugs that target these processes.