The effect of template RNA structure on elongation by HIV-1 reverse transcriptase
The effect of template RNA structure on elongation by HIV-1 reverse transcriptase
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DOI：
10.1016/s0167-4781(99)00011-1
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发表时间：
1999-03-19
期刊：
影响因子：
--
通讯作者：
Berkhout, B
中科院分区：
文献类型：
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作者：
Klasens, BIF;Huthoff, HT;Berkhout, B
逆转录病毒的RNA基因组的逆转录必须通过模板的一些高度结构化的区域进行。人类免疫缺陷病毒1型（HIV-1）的RNA基因组在病毒RNA的5'末端的重复（R）区域内含有两个发夹结构（图1）。这些结构，TAR和polyA发夹，在病毒生命周期中发挥重要作用。我们分析了HIV-1逆转录酶（RT）在野生型RNA模板及其突变体上的体外延伸特性，所述突变体具有稳定的或不稳定的polyA发夹。稳定的RNA结构被认为是干扰有效延长的RT酶，判断暂停cDNA产物的外观。一个直接的关系被测量之间的模板RNA结构的稳定性和RT暂停的程度。然而，结构诱导的暂停位点的位置是相当多样化的，在TAR和polyA发夹的碱基配对干的前面约6 nt的位置处具有显著的停止。这表明当RT酶的最前向结构域接触RNA双链体时，RT酶被停滞。发现将病毒核衣壳蛋白（NC）加入体外测定可克服这种结构诱导的RT停止。这些结果表明，RT聚合酶具有穿透具有稳定RNA结构的模板区域的问题。这种效应在高Mg 2+浓度下更明显，已知其稳定RNA二级结构。这种结构诱导的缺陷在病毒感染细胞中进行的逆转录测定中不明显，这是由NC蛋白或病毒粒子颗粒的其他组分引起的。因此，逆转录病毒可以使用相对稳定的RNA结构来控制病毒生命周期中的不同步骤，而不会干扰逆转录过程。(C)1999 Elsevier Science B. V.保留所有权利。
Reverse transcription of the RNA genome of retroviruses has to proceed through some highly structured regions of the template. The RNA genome of the human immunodeficiency virus type 1 (HIV-1) contains two hairpin structures within the repeat (R) region at the 5' end of the viral RNA (Fig. 1). These structures, the TAR and polyA hairpins, fulfil important functions in the viral life cycle. We analyzed the in vitro elongation properties of the HIV-1 reverse transcriptase (RT) enzyme on the wild-type RNA template and mutants thereof with either a stabilized or a destabilized polyA hairpin. Stable RNA structure was found to interfere with efficient elongation of the RT enzyme, as judged by the appearance of pause cDNA products. A direct relation was measured between the stability of template RNA structure and the extent of RT pausing. However, the position of structure-induced pause sites is rather diverse, with significant stops at a position approximately 6 nt ahead of the basepaired stem of the TAR and polyA hairpins. This suggests that the RT enzyme is stalled when its most forward domain contacts the RNA duplex. Addition of the viral nucleocapsid protein (NC) to the in vitro assay was found to overcome such structure-induced RT stops. These results indicate that the RT polymerase has problems penetrating regions of the template with stable RNA structure. This effect was more pronounced at high Mg2+ concentrations, which is known to stabilize RNA secondary structure. Such a structure-induced defect was not apparent in reverse transcription assays performed in virus-infected cells, which is either caused by the NC protein or other components of the virion particle. Thus, retroviruses can use relatively stable RNA structures to control different steps in the viral life cycle without interfering with the process of reverse transcription. (C) 1999 Elsevier Science B.V. All rights reserved.