Structural variability of the initiation complex of HIV-1 reverse transcription
Structural variability of the initiation complex of HIV-1 reverse transcription
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DOI：
10.1074/jbc.m404473200
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发表时间：
2004-08-20
影响因子：
4.8
通讯作者：
Marquet, R
中科院分区：
文献类型：
--
作者：
Goldschmidt, V;Paillart, JC;Marquet, R
HIV-1的逆转录是从与病毒RNA在引物结合位点（PBS）退火的tRNA（3）（Lys）分子开始的，但逆转录起始复合物的结构仍然存在争议。在这里，我们进行了原位结构探测，以及在体外的结构和功能的研究，由高度分歧的分离株（MAL和NL4.3/HXB 2）形成的起始复合物。我们的研究结果表明，起始复合物的结构是不保守的。在MAL中，根据14%的HIV-1分离株的序列分析，起始复合物的形成伴随着病毒RNA的复杂重排，并且需要与tRNA（3）（Lys）的广泛相互作用来有效启动逆转录。在NL4.3、HXB 2和大多数分离株中，tRNA（3）（Lys）退火对病毒RNA结构的影响最小，并且不需要PBS外的相互作用来实现逆转录的最佳起始。我们认为，在MAL中，需要与tRNA（3）（Lys）的广泛相互作用来驱动结构重排，从而产生最终被逆转录酶识别的结构元件。在NL4.3和HXB 2中，这些元件在tRNA（3）（Lys）退火之前已经存在于病毒RNA中，因此解释了不需要与引物的广泛相互作用。有趣的是，这种相互作用在设计为使用非同源tRNA作为引物（tRNA（His））的HXB 2突变体中是必需的。在后一种情况下，需要延长的相互作用来抵消与替代引物相关的负面贡献。
HIV-1 reverse transcription is initiated from a tRNA(3)(Lys) molecule annealed to the viral RNA at the primer binding site (PBS), but the structure of the initiation complex of reverse transcription remains controversial. Here, we performed in situ structural probing, as well as in vitro structural and functional studies, of the initiation complexes formed by highly divergent isolates (MAL and NL4.3/HXB2). Our results show that the structure of the initiation complex is not conserved. In MAL, and according to sequence analysis in 14% of HIV-1 isolates, formation of the initiation complex is accompanied by complex rearrangements of the viral RNA, and extensive interactions with tRNA(3)(Lys) are required for efficient initiation of reverse transcription. In NL4.3, HXB2, and most isolates, tRNA(3)(Lys) annealing minimally affects the viral RNA structure and no interaction outside the PBS is required for optimal initiation of reverse transcription. We suggest that in MAL, extensive interactions with tRNA(3)(Lys) are required to drive the structural rearrangements generating the structural elements ultimately recognized by reverse transcriptase. In NL4.3 and HXB2, these elements are already present in the viral RNA prior to tRNA(3)(Lys) annealing, thus explaining that extensive interactions with the primer are not required. Interestingly, such interactions are required in HXB2 mutants designed to use a non-cognate tRNA as primer (tRNA(His)). In the latter case, the extended interactions are required to counteract a negative contribution associate with the alternate primer.