Vo My-Nuong, Barany George, Rouzina Ioulia, Musier-Forsyth Karin
University of Minnesota, Department of Chemistry and Institute for Molecular Virology, Minneapolis, MN 55455, USA.
J Mol Biol. 2006 Oct 13;363(1):244-61. doi: 10.1016/j.jmb.2006.08.039. Epub 2006 Aug 22.
HIV-1 reverse transcription involves several nucleic acid rearrangements, which are catalyzed by the nucleocapsid protein (NC). Annealing of the trans-activation response element (TAR) DNA hairpin to a complementary TAR RNA hairpin, resulting in the formation of an extended 98-base-pair duplex, is an essential step in the minus-strand transfer step of reverse transcription. To elucidate the TAR RNA/DNA annealing reaction pathway, annealing kinetics were studied systematically by gel-shift assays performed in the presence or absence of HIV-1 NC. Truncated 27 nucleotide mini-TAR RNA and DNA constructs were used in this work. In the absence of NC, the annealing is slow, and involves the fast formation of an unstable extended "kissing" loop intermediate, followed by a slower strand exchange between the terminal stems. This annealing is very sensitive to loop-loop complementarity, as well as to nucleic acid concentration, ionic strength and temperature. NC stimulates the annealing approximately 5000-fold by stabilizing the bimolecular intermediate approximately 100 to 200-fold, and promoting the subsequent strand exchange reaction approximately 10 to 20-fold. NC concentration dependence studies suggest that there is a direct correlation between the amount of NC required to stabilize the intermediate and the amount needed to induce mini-TAR aggregation. Whereas saturating levels of NC are required to efficiently aggregate nucleic acids, sub-saturating NC is sufficient to significantly enhance duplex destabilization. Equilibrium levels of mini-TAR RNA/DNA annealing were also measured under a variety of conditions. Taken together, the results presented here provide a quantitative accounting of HIV-1 NC's aggregation and duplex destabilizing activity, and provide insights into the universal nucleic acid chaperone activity of this essential viral protein.
HIV-1逆转录涉及多种核酸重排，这些重排由核衣壳蛋白（NC）催化。反式激活应答元件（TAR）DNA发夹与互补的TAR RNA发夹退火，形成一个延伸的98个碱基对的双链体，这是逆转录负链转移步骤中的关键一步。为了阐明TAR RNA/DNA退火反应途径，通过在有或没有HIV-1 NC存在的情况下进行凝胶迁移分析，系统地研究了退火动力学。在这项工作中使用了截短的27个核苷酸的微型TAR RNA和DNA构建体。在没有NC的情况下，退火很慢，涉及不稳定的延伸“亲吻”环中间体的快速形成，随后是末端茎之间较慢的链交换。这种退火对环-环互补性以及核酸浓度、离子强度和温度非常敏感。NC通过将双分子中间体稳定约100至200倍，并将随后的链交换反应促进约10至20倍，从而将退火刺激约5000倍。NC浓度依赖性研究表明，稳定中间体所需的NC量与诱导微型TAR聚集所需的量之间存在直接相关性。虽然需要饱和水平的NC才能有效地聚集核酸，但亚饱和的NC足以显著增强双链体的去稳定作用。还在各种条件下测量了微型TAR RNA/DNA退火的平衡水平。综上所述，本文给出的结果对HIV-1 NC的聚集和双链体去稳定活性进行了定量描述，并为这种重要病毒蛋白的通用核酸伴侣活性提供了见解。
