Abstract
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.
Highlights
Chronic infection with hepatitis B virus (HBV) affects >250 million people worldwide, and the associated liver diseases cause up to 900,000 deaths per year [1]
Systematic evolution of ligands by exponential enrichment (SELEX) for duck HBV (DHBV) P binding RNAs yielded several DHBV ε RNA (Dε) variants with reduced base-pairing in the upper stem that remained primingproficient in vitro [40] and supported infection in vivo [41]; even marked variations in length and sequence of the upper Dε stem did not block in vitro priming and replication as long as a few sequence-determinants were present in accessible form [30]. From this we proposed a two-step priming activation model (Fig 1C, top left) [30,39] whereby initial P binding involves the bulge and its closing base-pairs, termed binding site 1 (BS1); a subsequent rearrangement in the apical stem enables the GUUGU motif to engage with P protein at BS2
As the appropriate extent of destabilization was unknown and previous studies had not revealed a systematic map of mutation-tolerant sites within ε [13,14,42,50,51] we employed a SELEX procedure to explore many ε variants in parallel for their ability to support HBV DNA formation in cells when part of a complete pgRNA
Summary
Chronic infection with hepatitis B virus (HBV) affects >250 million people worldwide, and the associated liver diseases cause up to 900,000 deaths per year [1]. The infectious cycle of all hepadnaviruses starts with envelope protein-mediated entry of virions into the target cell (for review: [7]), release of the nucleocapsid (core particle) into the cytoplasm, and delivery of the capsid-borne partially double-stranded (ds) relaxed circular (RC-) DNA into the nucleus; there it is converted, likely by host DNA repair factors [8], into covalently closed circular (ccc) DNA [9,10], the template for new viral transcripts These comprise subgenomic mRNAs for the envelope proteins and, in mammalian HBVs, for HBx, an epigenetic regulator of cccDNA transcriptional activity [11]; and the greater-than-genome length pgRNA (Fig 1A) plus the precore RNA from which the secretory hepatitis B e antigen (HBeAg) is derived [12].
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