Single Molecule HIV-1 Replication Interactions

单分子 HIV-1 复制相互作用

• 批准号: 9248371
• 负责人: MARK C WILLIAMS
• 金额: $ 38.04万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9248371
• 关键词: Affect; Anti-Retroviral Agents; Binding Proteins; Biochemical; Cells; DNA; DNA Binding; DNA Folding; DNA Probes; DNA Structure; Deaminase; Dimerization; Drug Targeting; Enzymes; Free Energy; Genomics; HIV-1; Human; Measurement; Measures; Mechanics; Methods; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; Nucleic Acid Probes; Nucleic Acids; Nucleocapsid; Nucleocapsid Proteins; Pharmaceutical Preparations; Process; Property; Proteins; RNA; RNA Folding; RNA-Directed DNA Polymerase; Regulation; Replication-Associated Process; Retrotransposon; Retroviridae; Reverse Transcription; Role; Single-Stranded DNA; Stretching; Structure; System; Testing; Viral; Work; design; dimer; drug resistant virus; ds-DNA; monomer; mutant; new therapeutic target; nucleic acid structure; protein structure; public health relevance; single molecule

 DESCRIPTION (provided by applicant): The objective of this work is to investigate the role of the HIV-1 nucleocapsid (NC) and reverse transcriptase (RT) proteins, as well as human APOBEC3 viral restriction factors, in the regulation of reverse transcription in retroviral systems The proposed work combines single molecule methods with biochemical methods and measurements in cells to obtain a complete understanding of nucleic acid (NA) interactions involved in retroviral replication. We use these methods to probe the mechanisms by which retroviral proteins dynamically restructure and organize NA to facilitate replication, and to determine how these processes are regulated. To do this, the PI has pioneered single molecule NA stretching methods that quantitatively probe NA structural rearrangements and protein-NA interactions. In the previous cycle, we demonstrated that the capability of NCs to rearrange NAs, referred to as NA chaperone activity, is directly correlated with HIV-1 replication in cells. The proposed work seeks to understand how this chaperone activity targets specific structures and facilitates NA reorganization without interfering with reverse transcription. In contrast to NC's facilitation of reverse transcription, human APOBEC3 (A3) proteins may inhibit reverse transcriptase activity. We will probe the DNA interactions of several A3 proteins and directly monitor RT activity with A3 proteins and NC. The specific aims are: (1) To determine how NC alters the conformational landscape of specific DNA and RNA structures. To test the hypothesis that HIV-1 NC is optimized to function as a chaperone with specific NA structures, we will directly measure the folding landscape of specific RNA and DNA structures in the absence and presence of wild type and mutant NC, determining the components of RNA and protein structure that optimize chaperone activity by lowering the unfolding barrier. (2) To probe the mechanism of APOBEC3 protein binding to single-stranded DNA. We hypothesize that different A3 proteins inhibit retroviral and retrotransposon replication both by acting as active enzymes that deaminate genomic sequences, as well as in a deaminase-independent manner. To test this hypothesis, we will probe the ssDNA binding activities of three representative A3 proteins: the primarily monomeric A3A, as well as wild type and mutant A3G and A3F, which both oligomerize. We will compare the results with HIV-1 replication inhibition by the same proteins in cells. (3) To mechanically measure the effects of HIV-1 NC and human APOBEC3 proteins on single DNA molecule elongation by HIV-1 reverse transcriptase. We will directly measure the elongation of single DNA molecules by RT in the absence and presence of wild type and mutant NC as well as A3 proteins with different oligomerization properties to determine the extent to which these protein enhance or inhibit RT activity.

 描述（由申请人提供）：这项工作的目的是研究 HIV-1 核衣壳 (NC) 和逆转录酶 (RT) 蛋白以及人 APOBEC3 病毒限制因子在逆转录调节中的作用逆转录病毒系统拟议的工作将单分子方法与生化方法和细胞测量相结合，以全面了解逆转录病毒复制中涉及的核酸（NA）相互作用。逆转录病毒蛋白动态重组和组织 NA 以促进复制，并确定这些过程是如何调节的，PI 开创了单分子 NA 拉伸方法，可定量探测 NA 结构重排和蛋白质-NA 相互作用。我们证明了 NC 重新排列 NA 的能力（称为 NA 伴侣活性）与细胞中的 HIV-1 复制直接相关。拟议的工作旨在了解这种伴侣活性如何针对特定结构并促进 NA 重组，而无需进行。与 NC 促进逆转录相反，人类 APOBEC3 (A3) 蛋白可能会抑制逆转录酶活性。我们将探测几种 A3 蛋白的 DNA 相互作用，并直接监测 A3 蛋白和 NC 的 RT 活性。 具体目标是：（1）为了确定 NC 如何改变特定 DNA 和 RNA 结构的构象景观，为了检验 HIV-1 NC 被优化为具有特定 NA 结构的伴侣的假设，我们将直接测量折叠。 (2)探讨APOBEC3蛋白与单链DNA结合的机制。我们寻找不同的 A3 蛋白通过充当基因组序列脱氨基的活性酶以及以不依赖脱氨酶的方式抑制逆转录病毒和逆转录转座子复制。为了检验这一假设，我们将探测三种代表性 A3 蛋白的 ssDNA 结合活性：主要单体 A3A 以及野生型和突变型 A3G 和 A3F，它们均寡聚化，我们将与细胞中相同蛋白质的 HIV-1 复制抑制作用进行比较。机械测量 HIV-1 NC 和人 APOBEC3 蛋白对 HIV-1 逆转录酶对单个 DNA 分子延伸的影响 我们将在野生型和突变型 NC 不存在和存在的情况下通过 RT 直接测量单个 DNA 分子的延伸。具有不同寡聚特性的 A3 蛋白，以确定这些蛋白增强或抑制 RT 活性的程度。