dNTP versus NTP discrimination by phenylalanine 451 in duck hepatitis B virus P protein indicates a common structure of the dNTP-binding pocket with other reverse transcriptases

Hepatitis B viruses, or hepadnaviruses, are small DNA-containing viruses that replicate through reverse transcription. Their prototype, HBV, causes severe liver disease in humans. The hepadnaviral P protein is an unusual reverse transcriptase (RT) that initiates DNA synthesis by host-factor-dependent protein priming on a specific RNA stem–loop template, ɛ, yielding a short DNA oligonucleotide covalently attached to the RT. This priming reaction can be reconstituted with in vitro-translated duck hepatitis B virus (DHBV) P protein. No direct structural data are available for any P protein. However, P proteins share a number of conserved motifs with other polymerases. Box A contains an invariant bulky residue recently shown to be crucial for dNTP versus NTP discrimination in RTs and some DNA polymerases; its equivalent in DHBV P protein would be phenylalanine 451 (F451). Four mutants, containing glycine (F451G), alanine (F451A), valine (F451V) and aspartate (F451D), were therefore analyzed for their ability to utilize dNTPs and NTPs in in vitro priming. Priming efficiencies with dNTPs decreased with decreasing side chain size but GTP utilization increased; the wild-type enzyme was inactive with GTP. In the context of complete DHBV genomes, all mutant proteins were competent for RNA encapsidation, indicating the absence of global structural alterations. Because the function of the discriminatory residue depends on its specific spatial disposition this strongly suggests a similar architecture for the P protein dNTP-binding pocket as in other RTs.

乙型肝炎病毒（Hepatitis B viruses，又称嗜肝DNA病毒Hepadnaviruses）是一类小型DNA病毒，通过逆转录（reverse transcription）方式完成复制。其原型株为HBV，可引发人类重症肝脏疾病。嗜肝DNA病毒P蛋白是一类特殊的逆转录酶（reverse transcriptase, RT），它通过宿主因子依赖的蛋白引发机制，在特定RNA茎环模板ɛ上启动DNA合成，生成一段与逆转录酶共价结合的短DNA寡核苷酸。该引发反应可通过体外翻译的鸭乙型肝炎病毒（duck hepatitis B virus, DHBV）P蛋白实现体外重建。目前尚无任何P蛋白的直接结构数据。不过，P蛋白与其他聚合酶共享多个保守基序。A框（Box A）包含一个保守的大体积残基，近期研究证实该残基在逆转录酶及部分DNA聚合酶的dNTP与NTP区分过程中发挥关键作用；该残基在鸭乙型肝炎病毒P蛋白中的对应位点为苯丙氨酸451（F451）。为此，研究人员构建了4种突变体，分别为F451G、F451A、F451V及F451D，并分析它们在体外引发反应中利用dNTP与NTP的能力。结果显示，以dNTP为底物时的引发效率随侧链体积减小而降低，但对GTP的利用效率却有所提升；野生型酶无法利用GTP。在完整鸭乙型肝炎病毒基因组背景下，所有突变体蛋白均具备RNA衣壳化能力，表明其未发生全局结构改变。由于该区分残基的功能依赖于其特定空间排布，这一结果强烈提示P蛋白的dNTP结合口袋与其他逆转录酶具有相似的结构特征。