Kim J K, Palaniappan C, Wu W, Fay P J, Bambara R A
Department of Biochemistry and Biophysics, University of Rochester, Rochester, New York 14642, USA.
J Biol Chem. 1997 Jul 4;272(27):16769-77. doi: 10.1074/jbc.272.27.16769.
We previously found that strand transfer by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is promoted at sites where RT pauses during synthesis. In this report, strand transfer is measured within the 5' transactivation response region (TAR) of HIV-1 RNA. We hypothesized that the stable hairpin structure of TAR would induce RT pausing, promoting RNase H-directed cleavage of the template and subsequent transfer at that site. We further predicted that HIV-1 nucleocapsid protein (NC), known to melt secondary structures, would decrease transfer. We show that TAR created a strong pause site for RT, but NC significantly promoted strand transfer. The effect of NC is specific, since other single strand binding proteins failed to stimulate transfer. In another unexpected outcome, preferred positions of internal transfer were not at the pause site but were in the upper stem and loop of TAR. Thus, we propose a new mechanism for transfer within TAR described by an interactive hairpin model, in which association between the donor and the acceptor templates within the TAR stem promotes transfer. The model is consistent with the observed stimulation of strand transfer by NC. The model is applicable to internal and replicative end transfer.
我们先前发现，人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）的链转移在合成过程中RT暂停的位点处会得到促进。在本报告中，我们测定了HIV-1 RNA的5'反式激活应答区域（TAR）内的链转移。我们推测，TAR稳定的发夹结构会诱导RT暂停，促进核糖核酸酶H介导的模板切割以及随后在该位点的转移。我们进一步预测，已知可解开二级结构的HIV-1核衣壳蛋白（NC）会减少转移。我们发现，TAR为RT创造了一个强烈的暂停位点，但NC显著促进了链转移。NC的作用具有特异性，因为其他单链结合蛋白未能刺激转移。另一个意外的结果是，内部转移的优先位置不在暂停位点，而是在TAR的上部茎环结构中。因此，我们提出了一种由交互式发夹模型描述的TAR内转移的新机制，其中TAR茎内供体模板和受体模板之间的结合促进了转移。该模型与观察到的NC对链转移刺激作用一致。该模型适用于内部转移和复制性末端转移。
