Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Freiburg, Germany.
PLoS Pathog. 2022 Mar 8;18(3):e1010362. doi: 10.1371/journal.ppat.1010362. eCollection 2022 Mar.
Hepadnaviruses, including hepatitis B virus (HBV) as a major human pathogen, replicate their tiny 3 kb DNA genomes by capsid-internal protein-primed reverse transcription of a pregenomic (pg) RNA. Initiation requires productive binding of the viral polymerase, P protein, to a 5´ proximal bipartite stem-loop, the RNA encapsidation signal ε. Then a residue in the central ε bulge directs the covalent linkage of a complementary dNMP to a Tyr sidechain in P protein´s Terminal Protein (TP) domain. After elongation by two or three nucleotides (nt) the TP-linked DNA oligo is transferred to a 3´ proximal acceptor, enabling full-length minus-strand DNA synthesis. No direct structural data are available on hepadnaviral initiation complexes but their cell-free reconstitution with P protein and ε RNA (Dε) from duck HBV (DHBV) provided crucial mechanistic insights, including on a major conformational rearrangement in the apical Dε part. Analogous cell-free systems for human HBV led at most to P-ε binding but no detectable priming. Here we demonstrate that local relaxation of the highly basepaired ε upper stem, by mutation or via synthetic split RNAs, enables ε-dependent in vitro priming with full-length P protein from eukaryotic translation extract yet also, and without additional macromolecules, with truncated HBV miniP proteins expressed in bacteria. Using selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) we confirm that upper stem destabilization correlates with in vitro priming competence and show that the supposed bulge-closing basepairs are largely unpaired even in wild-type ε. We define the two 3´ proximal nt of this extended bulge as main initiation sites and provide evidence for a Dε-like opening of the apical ε part upon P protein binding. Beyond new HBV-specific basic aspects our novel in vitro priming systems should facilitate the development of high-throughput screens for priming inhibitors targeting this highly virus-specific process.
嗜肝 DNA 病毒，包括作为主要人类病原体的乙型肝炎病毒 (HBV)，通过衣壳内蛋白引发的前基因组 (pg) RNA 的反转录来复制其微小的 3 kb DNA 基因组。起始需要病毒聚合酶 P 蛋白与 5'近端二部分茎环，即 RNA 衣壳信号 ε 进行有效结合。然后，ε 环中部的一个残基指导互补的 dNMP 与 P 蛋白的末端蛋白 (TP) 结构域中的 Tyr 侧链共价连接。延伸两个或三个核苷酸 (nt) 后，TP 连接的 DNA 寡核苷酸转移到 3'近端受体，从而能够进行全长负链 DNA 的合成。目前尚无嗜肝 DNA 病毒起始复合物的直接结构数据，但它们与来自鸭乙型肝炎病毒 (DHBV) 的 P 蛋白和 ε RNA (Dε) 的无细胞重建提供了至关重要的机制见解，包括在 ε 上部结构域的主要构象重排。类似的人类 HBV 无细胞系统最多只能导致 P-ε 结合，但不能检测到引发。在这里，我们证明通过突变或通过合成分裂 RNA 局部放松高度碱基配对的 ε 上部茎，可以使 ε 依赖的全长 P 蛋白在真核翻译提取物中进行体外引发，同时也可以在没有额外大分子的情况下，使用在细菌中表达的截断 HBV miniP 蛋白进行体外引发。我们使用选择性 2-羟基乙酰化分析通过引物延伸 (SHAPE) 证实了上茎的不稳定与体外引发能力相关，并表明即使在野生型 ε 中，假定的环关闭碱基对也大部分未配对。我们将这个扩展环的两个 3'近端 nt 定义为主要起始位点，并提供了 P 蛋白结合后 ε 上部结构域类似 Dε 打开的证据。除了新的 HBV 特异性基本方面外，我们的新型体外引发系统应该有助于开发针对这种高度病毒特异性过程的引发抑制剂的高通量筛选。
