부산대학교 도서관

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. Author summary: Chronic hepatitis B virus (HBV) infection puts >250 million people at an increased risk for severe liver disease. Current treatments can control but rarely cure infection. HBV features a 3,200 bp DNA genome, generated by reverse transcription of a pregenomic (pg) RNA. To initiate DNA synthesis the viral polymerase, P protein, employs a stem-loop on pgRNA, ε, to covalently link a defined first nucleotide to its Terminal Protein (TP) domain. This protein-priming is highly virus-specific yet poorly understood. More is known for duck HBV (DHBV) where, different from HBV, protein-priming was successfully reconstituted in vitro years ago. One insight was that gaining priming-competence involves opening of the apical stem in DHBV ε RNA (Dε); in HBV ε the more extensive basepairing might restrict such dynamics. Here we relaxed these constraints by identifying functional but less stably folded, including split, HBV ε variants. Several such variants supported in vitro priming, including in a simple two-component-system employing a shortened recombinant P protein. Amongst other data the new cell-free systems yielded a first view on a major conformational change in HBV ε RNA bound to P protein, highlighting the importance of RNA dynamics for the human virus. Beyond furthering basic understanding our data should facilitate screening for protein-priming inhibitors as new anti-HBV agents. [ABSTRACT FROM AUTHOR]