Single-Molecule Spectroscopy of HIV-1 Replication Complexes

几种核蛋白复合物的单分子光谱研究表明，HIV-1逆转录酶（RT）根据核酸底物的性质具有不同的取向。一个意想不到的结果是，我们观察到非核苷类RT抑制剂（NNRTIs）诱导酶与多嘌呤道（PPT）的结合方向与DNA合成的起始方向不相容。随后的研究检查了trna引发的（-）链DNA合成的起始，表明碱基配对的U5-IR干位于引物结合位点的下游，控制着酶的取向，并且可以通过包括HIV-1核衣壳（NC）蛋白来减轻这种起始障碍。我们正在继续用单分子光谱研究含nc的Gag前体和RT突变对以下方面的影响：(a) HIV-1基因组上trna引物起始，(b)相关慢病毒rna上tRNALys、3引物起始，(c)中央终止序列（CTS）终止动力学，(d)伽玛逆转录病毒RT的构象动力学。这些项目结合了通过Click化学在含有AZ-Phe的RT亚基中特定位点放置荧光团的新策略。

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.