Intrinsic Nucleic Acid Dynamics Modulates HIV-1 Nucleocapsid Protein Binding to Its Targets

HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using 13C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome.

HIV-1型核衣壳蛋白（NC）参与逆转录关键步骤中的核酸重排过程。该蛋白通过其两个锌指结构，优先与单链序列中的未配对鸟嘌呤发生相互作用。在对应于HIV-1基因组反式激活应答元件（transactivation response element, TAR）的互补脱氧核糖核酸（complementary DNA, cDNA）拷贝上半部分的迷你cTAR茎环结构中，研究发现NC对该迷你cTAR茎底部的TGG序列具有显著偏好性。为进一步阐明该结合位点如何在多个含未配对鸟嘌呤的潜在结合位点中被筛选出来，我们通过碳-13弛豫测量探究了迷你cTAR的内在动力学特性。我们采用无模型分析法对自旋弛豫时间的测量结果进行了分析，并辅以弛豫弥散测量进行补充。我们的数据表明，茎部下方被优先识别的鸟嘌呤不存在构象交换现象，且具有较高的运动性。与之相反，迷你cTAR中未被识别的未配对鸟嘌呤则存在构象交换现象，这可能与瞬时碱基配对有关。上述研究结果支持这一观点：NC可优先识别具有高运动性的未配对鸟嘌呤。NC通过动力学特性区分相近序列的能力，有助于阐释NC如何识别HIV基因组内的特定结合位点。