Single-Molecule Spectroscopy of HIV-1 Replication Complexes

HIV-1 复制复合物的单分子光谱

• 批准号: 9343931
• 负责人: Stuart F. J. Le Grice
• 金额: $ 22.34万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9343931
• 关键词: Base Pairing; Binding; Binding Sites; Chemistry; Complex; DNA biosynthesis; Enzymes; Gammaretrovirus; Genome; HIV-1; Lysine-Specific tRNA; Mediating; Mutation; Nature; Nucleic Acids; Nucleocapsid; Nucleocapsid Proteins; Nucleoproteins; Outcome; RNA-Directed DNA Polymerase; Research; Retrotransposon; Site; Spectrum Analysis; Transfer RNA; fluorophore; mutant; non-nucleoside reverse transcriptase inhibitors; single molecule; stem; viral RNA

Single-molecule spectroscopy studies of several nucleoprotein complexes demonstrated that HIV-1 reverse transcriptase (RT) assumes different orientations, depending on the nature of the nucleic acid substrate. An unexpected outcome was our observation that nonnucleoside RT inhibitors (NNRTIs) induce enzyme binding to the polypurine tract (PPT) in an orientation incompatible with initiating DNA synthesis. Subsequent studies examined tRNA-primed initiation of (-) strand DNA synthesis, showing that the base-paired U5-IR stem immediately downstream of the primer binding site controls enzyme orientation and that this barrier to initiation can be alleviated by including the HIV-1 nucleocapsid (NC) protein. We are continuing single-molecule spectroscopy to examine the effect of NC-containing Gag precursors and RT mutations on (a) tRNA-primed initiation on the HIV-1 genome, (b) tRNALys,3-primed initiation on related lentiviral RNAs, (c) dynamics of termination at the central termination sequence (CTS), and (d) conformational dynamics of a gammaretroviral RT. These projects are combined with new strategies for site-specific placement of fluorophores within RT subunits containing AZ-Phe via Click chemistry.

几种核蛋白复合物的单分子光谱研究表明，根据核酸底物的性质，HIV-1逆转录酶（RT）假定不同方向。一个意外的结果是，我们的观察结果是，非核苷RT抑制剂（NNRTIS）诱导酶结合与息肉素裂纹（PPT）的结合在与启动DNA合成不符的方向中。随后的研究检查了（ - ）链DNA合成的tRNA引发的启动，表明底漆结合位点的碱基对U5-ir茎立即在下游下游控制酶方向，并且可以通过包括HIV-1核糖质（NC）蛋白质来缓解这种启动的障碍。我们正在继续进行单分子光谱检查，以检查含NC的GAG前体和RT突变对HIV-1基因组的（a）tRNA引发的启动的影响，（b）TRNALYS，3个序列对相关慢病毒RNA的启动对中央终止序列序列（CT）和（CT）的动力学（CT）和（CT）的动力学（CT）和（c）的动力学（CT），（CT）的动态（c）这些项目与通过点击化学中含有AZ-PHE的RT亚基中特定地点的荧光团特定位置相结合。