SELECTED MUTATIONS OF THE DUCK HEPATITIS-B VIRUS-P GENE RNASE-H DOMAIN AFFECT BOTH RNA PACKAGING AND PRIMING OF MINUS-STRAND DNA-SYNTHESIS
SELECTED MUTATIONS OF THE DUCK HEPATITIS-B VIRUS-P GENE RNASE-H DOMAIN AFFECT BOTH RNA PACKAGING AND PRIMING OF MINUS-STRAND DNA-SYNTHESIS
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DOI：
10.1128/jvi.68.8.5232-5238.1994
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发表时间：
1994-08-01
影响因子：
5.4
通讯作者：
MARION, PL
中科院分区：
文献类型：
--
作者：
CHEN, Y;ROBINSON, WS;MARION, PL
所有嗜肝DNA病毒的基因组都有一个称为P基因的开放阅读框，它编码90至97 kDa的多肽。该P基因的一个或多个产物参与病毒生命周期的多种功能。这些功能包括启动负链 DNA 合成的引发活性、使用 RNA 或 DNA 模板（逆转录酶）合成 DNA 的聚合酶活性、降解 RNA-DNA 杂交体的 RNA 链的核酸酶活性 (RNase H) 以及参与将 RNA 前基因组包装成核壳。在之前的研究中，我们发现鸭乙型肝炎病毒（DHBV）P基因产物RNase H结构域711位的单点突变阻止了病毒RNA包装。在本实验中，我们对 DHBV RNase H 结构域中的额外保守氨基酸进行了突变，并检查了包含这些突变的病毒基因组包装 RNA 和复制病毒 DNA 的能力。与 Cys-711 相邻的带电氨基酸和硫基氨基酸发生突变。这些突变体在 RNA 包装或病毒复制方面均不存在缺陷。我们还根据大肠杆菌和人类免疫缺陷病毒逆转录酶 RNase H 酶的晶体结构中的共同元件以及它们的氨基酸序列与 DHBV 和 HBV 的 RNase H 结构域的相似性，测试了许多突变。我们的结果表明，整个β4链以及β4、αA和αB区域的假定疏水核心中的氨基酸Leu-712、Leu-697和Val-719分别参与前基因组RNA衣壳化。这表明 DHBV P 基因产物中 RNase H 结构域的基本结构是病毒 RNA 包装所必需的。我们使用 Wang 和 Seeger 开发的体外 DHBV 负链 DNA 引发系统（G.-H. Wang 和 C. Seeger, Cell 71:663-670, 1992）来测试 RNase H 包装突变对 P 基因产物酶活性的影响。虽然所有测试的包装缺陷突变体都保持了 DNA 引发活性，但与野生型基因组相比，其水平降低了 5 至 20 倍。这一观察结果表明嗜肝DNA病毒RNase H结构域在优化负链DNA合成的引发中发挥着作用。
The genome of all hepadnaviruses has an open reading frame called the P gene, which encodes a polypeptide of 90 to 97 kDa. The product or products of this P gene are involved in multiple functions of the viral life cycle. These functions include a priming activity which initiates minus-strand DNA synthesis, a polymerase activity which synthesizes DNA by using either RNA or DNA templates (reverse transcriptase), a nuclease activity which degrades the RNA strand of RNA-DNA hybrids (RNase H), and involvement in packaging the RNA pregenome into nucleocaspsids. In a previous study, we found that a single point mutation at position 711 in the duck hepatitis B virus (DHBV) P gene product RNase H domain prevented viral RNA packaging. In the present experiments, we have mutated additional conserved amino acids in the DHBV RNase H domain and examined the ability of viral genomes containing these mutations to package RNA and replicate viral DNA. Charged and sulfur group amino acids adjacent to Cys-711 were mutated. None of these mutants was defective in either RNA packaging or viral replication. We also tested a number of mutations on the basis of common elements in the crystal structures of Escherichia coli and human immunodeficiency virus reverse transcriptase RNase H enzymes and on the basis Of the similarities df their amino acid sequences to those of the RNase H domains of DHBV and HBV. Our results revealed that the entire beta 4 strand and amino acids Leu-712, Leu-697, and Val-719 in the putative hydrophobic cores of the beta 4, alpha A, and alpha B regions, respectively, are involved in pregenomic RNA encapsidation. This suggests that the basic structure of the RNase H domain in the DHBV P gene product is required for viral RNA packaging. We used the in vitro DHBV minus-strand DNA priming system developed by Wang and Seeger (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992) to test the effect of RNase H packaging mutations on P gene product enzymatic activity. While all packaging-defective mutants tested maintained DNA priming activity, levels were decreased 5- to 20-fold compared with that of the wild-type genome. This observation suggests that the hepadnavirus RNase H domain plays a role in optimizing priming of minus-strand DNA synthesis.