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（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APOBEC 3病毒限制因子的作用，在逆转录病毒系统中逆转录的调节中所提出的工作将单分子方法与生物化学方法和细胞测量相结合，以获得对核酸（NA）的完整理解。参与逆转录病毒复制的相互作用。我们使用这些方法来探测逆转录病毒蛋白动态重组和组织NA以促进复制的机制，并确定这些过程是如何调节的。为此，PI开创了单分子NA拉伸方法，定量探测NA结构重排和蛋白质-NA相互作用。在前一个周期中，我们证明了NC重排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分子伴侣活性如何靶向特定的结构，并促进NA重组，而不干扰逆转录。与NC促进逆转录相反，人APOBEC 3（A3）蛋白可抑制逆转录酶活性。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）确定NC如何改变特定DNA和RNA结构的构象景观。为了验证HIV-1 NC被优化为具有特定NA结构的伴侣蛋白的假设，我们将在不存在和存在野生型和突变NC的情况下直接测量特定RNA和DNA结构的折叠景观，确定RNA的组分和蛋白质结构通过降低解折叠屏障来优化伴侣蛋白活性。(2)探讨APOBEC 3蛋白与单链DNA结合的机制。我们假设，不同的A3蛋白抑制逆转录病毒和逆转录转座子复制作为活性酶，脱氨基因组序列，以及在脱氨酶独立的方式。为了验证这一假设，我们将探测三种代表性A3蛋白的ssDNA结合活性：主要是单体A3 A，以及野生型和突变型A3 G和A3 F，两者都寡聚化。我们将比较结果与细胞中相同蛋白质对HIV-1复制的抑制作用。(3)机械测量HIV-1 NC和人APOBEC 3蛋白对HIV-1逆转录酶延伸单个DNA分子的影响。我们将在不存在和存在野生型和突变NC以及具有不同寡聚化性质的A3蛋白的情况下通过RT直接测量单个DNA分子的延伸，以确定这些蛋白增强或抑制RT活性的程度。