Single Molecule HIV-1 Replication Interactions

单分子 HIV-1 复制相互作用

• 批准号: 9095336
• 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/9095336
• 关键词: Affect; Anti-Retroviral Agents; Binding Proteins; Biochemical; Cells; DNA; DNA Binding; DNA Folding; DNA Probes; DNA Structure; Deaminase; Drug Targeting; Enzymes; Free Energy; Genomics; HIV-1; Health; Human; Measurement; Measures; Methods; Molecular; Molecular Chaperones; 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; 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APOBEC3病毒限制因子在逆转录病毒系统中逆转录调控中的作用。建议的工作结合单分子方法与生化方法和细胞测量，以获得对逆转录病毒复制中涉及的核酸(NA)相互作用的完整了解。我们使用这些方法来探索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NCS重排Nas的能力，称为NA伴侣活性，与HIV-1在细胞中的复制直接相关。这项拟议的工作试图了解这种伴侣活性如何针对特定的结构，并在不干扰逆转录的情况下促进NA重组。与NC促进逆转录相反，人APOBEC3(A3)蛋白可能会抑制逆转录酶的活性。我们将探索几个A3蛋白的DNA相互作用，并直接监测与A3蛋白和NC的RT活性。 具体目的是：(1)确定NC如何改变特定DNA和RNA结构的构象格局。为了验证HIV-1 NC被优化为具有特定NA结构的伴侣的假设，我们将在没有野生型和突变NC的情况下直接测量特定RNA和DNA结构的折叠景观，确定通过降低展开屏障来优化伴侣活性的RNA和蛋白质结构的成分。(2)探讨APOBEC3蛋白与单链DNA结合的机制。我们假设，不同的A3蛋白通过作为脱氨酶的活性酶来抑制逆转录病毒和逆转录转座子的复制，以及以脱氨酶非依赖的方式发挥作用。为了验证这一假设，我们将探索三个具有代表性的A3蛋白的单链DNA结合活性：主要的单体A3A，以及野生型和突变型A3G和A3F，它们都是寡聚体。我们将把结果与细胞中相同蛋白质抑制HIV-1复制的结果进行比较。(3)机械检测HIV-1NC蛋白和人APOBEC3蛋白对HIV-1逆转录酶单DNA分子伸长的影响。我们将在野生型和突变型NC以及具有不同寡聚性质的A3蛋白的存在和不存在的情况下，通过RT直接测量单个DNA分子的伸长，以确定这些蛋白增强或抑制RT活性的程度。