Single Molecule HIV-1 NC/Gag-DNA Interactions

单分子 HIV-1 NC/Gag-DNA 相互作用

基本信息

批准号：
7927901
负责人：
MARK C WILLIAMS
金额：
$ 30.49万
依托单位：
NORTHEASTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7927901
关键词：
Binding Biological Assay Cells DNA DNA Binding DNA Probes DNA Sequence Rearrangement Development Dimerization Drug Delivery Systems Elements Exhibits Feline Immunodeficiency Virus Gagging HIV HIV-1 HIV-1 Reverse Transcriptase Human Human T-lymphotropic virus 1 Inositol Phosphates Kinetics Lead Measurement Measures Methods Molecular Molecular Chaperones Moloney Leukemia Virus Nucleic Acid Binding Nucleic Acid Probes Nucleic Acids Nucleocapsid Nucleocapsid Proteins Play Process Property Proteins Publishing RNA RNA-Directed DNA Polymerase Regulation Research Retrotransposition Retrotransposon Reverse Transcription Role Rous sarcoma virus Single-Stranded DNA Staging Stretching Structure System Tertiary Protein Structure Testing Thermodynamics Variant Viral Viral Reverse Transcription Virus-like particle Work ds-DNA human CEM15 protein in vivo mutant novel polymerization public health relevance research study response self assembly single molecule viral RNA

项目摘要

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.

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变和病毒自组装的过程调节中的作用。该 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方法来测试有关核酸-蛋白质相互作用的特定假设，这对于逆转录病毒包装和复制很重要。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 伴侣活性）对于逆转录病毒复制至关重要。 NA 拉伸方法特别适合探测 NA 伴侣活性，因为可以直接控制核酸结构重排和包装，并且可以量化所得反应。将开发一种新方法来机械地探测逆转录并表征对 HIV-1 复制重要的蛋白质在此过程中的影响。具体目标是：（1）量化野生型和突变型HIV-1以及其他逆转录病毒核酸伴侣蛋白与单链和双链DNA以及特定RNA结构的相互作用。这项工作将检验核酸伴侣活性由三个关键要素组成的假设：核酸聚集、核酸去稳定和快速蛋白质-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。我们假设 APOBEC 蛋白通过改变特定的 DNA 相互作用来抑制逆转录。为了检验这一假设，我们将检查人类 APOBEC 与单链和双链 DNA 相互作用的热力学和动力学。 (3)量化野生型和突变型HIV-1 Gag的核酸相互作用。尽管 HIV-1 NC 是 Gag 的结构域，主要负责与 NA 的相互作用，但这些蛋白质促进了细胞中显着不同的过程。该目标旨在了解 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 APOBEC3G 存在的情况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，这反过来又将有助于开发针对这些过程的药物。