Medical Center-University of Freiburg, Department of Internal Medicine II/Molecular Biology, University of Freiburg, 79106 Freiburg, Germany;
Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120 Heidelberg, Germany.
Proc Natl Acad Sci U S A. 2021 Mar 30;118(13). doi: 10.1073/pnas.2022373118.
Hepadnaviruses, with the human hepatitis B virus as prototype, are small, enveloped hepatotropic DNA viruses which replicate by reverse transcription of an RNA intermediate. Replication is initiated by a unique protein-priming mechanism whereby a hydroxy amino acid side chain of the terminal protein (TP) domain of the viral polymerase (P) is extended into a short DNA oligonucleotide, which subsequently serves as primer for first-strand synthesis. A key component in the priming of reverse transcription is the viral RNA element epsilon, which contains the replication origin and serves as a template for DNA primer synthesis. Here, we show that recently discovered non-enveloped fish viruses, termed nackednaviruses [C. Lauber , 22, 387-399 (2017)], employ a fundamentally similar replication mechanism despite their huge phylogenetic distance and major differences in genome organization and viral lifestyle. In vitro cross-priming studies revealed that few strategic nucleotide substitutions in epsilon enable site-specific protein priming by heterologous P proteins, demonstrating that epsilon is functionally conserved since the two virus families diverged more than 400 Mya. In addition, other elements crucial for the hepadnavirus-typical replication of pregenomic RNA into relaxed circular double-stranded DNA were identified at conserved positions in the nackednavirus genomes. Hence, the replication mode of both hepadnaviruses and nackednaviruses was already established in their Paleozoic common ancestor, making it a truly ancient and evolutionary robust principle of genome replication that is more widespread than previously thought.
嗜肝 DNA 病毒，以人类乙型肝炎病毒为原型，是小的、包膜的嗜肝 DNA 病毒，通过 RNA 中间体的逆转录进行复制。复制是通过独特的蛋白引发机制启动的，病毒聚合酶（P）的末端蛋白（TP）结构域中的一个羟基氨基酸侧链延伸成一个短的 DNA 寡核苷酸，随后作为第一链合成的引物。逆转录引发的一个关键组成部分是病毒 RNA 元件 ε，它包含复制起点，并作为 DNA 引物合成的模板。在这里，我们表明，最近发现的无包膜鱼类病毒，称为 nackednaviruses [C. Lauber, 22, 387-399 (2017)]，尽管在系统发育上距离很远，基因组组织和病毒生活方式存在重大差异，但它们采用了基本相似的复制机制。体外交叉引发研究表明，ε 中的少数战略核苷酸取代使异源 P 蛋白能够进行特异性蛋白引发，这表明 ε 在功能上是保守的，因为这两个病毒家族在 4 亿多年前就已经分化。此外，在 nackednavirus 基因组中保守位置鉴定出其他对前基因组 RNA 松弛环状双链 DNA 典型复制至关重要的 元件。因此，嗜肝 DNA 病毒和 nackednaviruses 的复制模式在它们的古生代共同祖先中就已经建立，这使得它成为一种真正古老且进化稳健的基因组复制原则，比以前想象的更为广泛。
