SELEX-derived aptamers of the duck hepatitis B virus RNA encapsidation signal distinguish critical and non-critical residues for productive initiation of reverse transcription
SELEX-derived aptamers of the duck hepatitis B virus RNA encapsidation signal distinguish critical and non-critical residues for productive initiation of reverse transcription
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DOI：
10.1093/nar/gkh772
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发表时间：
2004-08-01
影响因子：
14.9
通讯作者：
Nassal, M
中科院分区：
文献类型：
--
作者：
Hu, KH;Beck, J;Nassal, M
B型肝炎病毒（HBV）的蛋白质引发复制是通过逆转录酶（P蛋白）与前基因组RNA上凸出的茎环的伴侣依赖性结合以及第一DNA链的5'末端核苷酸的ε模板合成而启动的。P蛋白如何识别起始位点还知之甚少。在哺乳动物HBV和鸭HBV（DHBV）中，整个茎环是广泛碱基配对的;在其他禽类HBV中，茎上部区域具有低碱基配对潜力。DHBV体外翻译后可重建起始点，但HBV、P蛋白和DHBV依赖性RNA不能重建起始点。采用SELEX方法对Dependant上茎变体的限制性文库进行筛选，获得了一系列结合良好的适体。大多数含有C-丰富的共识基序，具有非常低的碱基配对潜力，一些支持启动，其他没有。基于拟南芥的次级突变体允许将这种功能差异固定在保守环侧翼的残基上，并且未配对的U。体外活性共有序列也支持病毒复制。因此，上部茎的大部分充当间隔区，如果不是碱基配对，则保证相关锚残基的可接近性。这表明，碱基配对的依赖性代表了一个例外，而不是一个原型的禽HBV的依赖性信号，它提供了一个解释，为什么尝试在体外重建启动与人HBV迄今失败。
Protein-primed replication of hepatitis B viruses (HBVs) is initiated by the chaperone dependent binding of the reverse transcriptase (P protein) to the bulged epsilon stem-loop on the pregenomic RNA, and the epsilon-templated synthesis of the 5' terminal nucleotides of the first DNA strand. How P protein recognizes the initiation site is poorly understood. In mammalian HBVs and in duck HBV (DHBV) the entire stem-loop is extensively base paired; in other avian HBVs the upper stem regions have a low base pairing potential. Initiation can be reconstituted with in vitro translated DHBV, but not HBV, P protein and DHBV epsilon (Depsilon) RNA. Employing the SELEX method on a constrained library of Depsilon upper stem variants, we obtained a series of well-binding aptamers. Most contained C-rich consensus motifs with very low base pairing potential; some supported initiation, others did not. Consensus-based secondary mutants allowed to pin down this functional difference to the residues flanking the conserved loop, and an unpaired U. In vitro active consensus sequences also supported virus replication. Hence, most of the upper stem acts as a spacer, which, if not base paired, warrants accessibility of relevant anchor residues. This suggests that the base paired Depsilon represents an exceptional rather than a prototypic avian HBV epsilon signal, and it offers an explanation as to why attempts to in vitro reconstitute initiation with human HBV have thus far failed.