In all hepadnaviruses, protein-primed reverse transcription of the pregenomic RNA (pgRNA) is initiated by binding of the viral polymerase, P protein, to the RNA element. Universally, consists of a lower stem and an upper stem, separated by a bulge, and an apical loop. Complex formation triggers pgRNA encapsidation and the -templated synthesis of a DNA oligonucleotide (priming) that serves to generate minus-strand DNA. In vitro systems for duck hepatitis B virus (DHBV) yielded important insights into the priming mechanism, yet their relevance in infection is largely unexplored. Moreover, additional functions encoded in the DHBV (D) sequence could affect in vivo fitness. We therefore assessed the in vivo performances of five recombinant DHBVs bearing multiple mutations in the upper D stem. Three variants with only modestly reduced in vitro replication competence established chronic infection in ducks. From one variant but not another, three adapted new variants emerged upon passaging, as demonstrated by increased relative fitness in coinfections with wild-type DHBV. All three showed enhanced priming and replication competence in vitro, and in one, DHBV e antigen (DHBeAg) production was restored. Pronounced impacts on other D functions were not detected; however, gradual, synergistic contributions to overall performance are suggested by the fact of none of the variants reaching the in vivo fitness of wild-type virus. These data shed more light on the P-D interaction, define important criteria for the design of future in vivo evolution experiments, and suggest that the upper D stem sequences provided an evolutionary playground for DHBV to optimize in vivo fitness.Hepadnaviruses are small enveloped hepatotropic DNA viruses that replicate through protein-primed reverse transcription (for reviews, see references 8 and 38). Human hepatitis B virus (HBV) and duck HBV (DHBV) are the respective prototypes of the mammalian and avian HBVs, which share a similar genome organization and replication strategy (Fig. 1A). Their ϳ3-kb genomes encode a single core protein, three or two (avian viruses) envelope proteins, an unusual reverse transcriptase (P protein), and, only in the mammalian viruses, the poorly understood X protein. Virions contain the genome as a relaxed circular DNA (rcDNA), which upon infection is converted into nuclear covalently closed circular DNA (cccDNA), the template for all viral transcripts. In addition to the subgenomic RNAs (sgRNAs), two types of greater-than-genomelength RNAs are produced. The precore RNAs serve for translation of the precursors of the secretory hepatitis B virus e antigens (HBeAg and DHBeAg, respectively), which may modulate the host's immune response but are nonessential. The pregenomic RNAs (pgRNAs), by contrast, are vital as mRNAs for core and P protein and by constituting the obligate template for new DNA genomes.Reverse transcription begins with specific packaging of pgRNA into newly forming capsids, triggered by binding of P protein to the 5Ј-proximal ε (Dε for DHBV) stem-loop (...
Hepadnaviruses, including human hepatitis B virus (HBV), replicate their tiny DNA genomes by protein-primed reverse transcription of a pregenomic (pg) RNA. Replication initiation as well as pgRNA encapsidation depend on the interaction of the viral polymerase, P protein, with the ε RNA element, featuring a lower and an upper stem, a central bulge, and an apical loop. The bulge, somehow assisted by the loop, acts as template for a P protein-linked DNA oligo that primes full-length minus-strand DNA synthesis. Phylogenetic conservation and earlier mutational studies suggested the highly based-paired ε structure as crucial for productive interaction with P protein. Using the tractable duck HBV (DHBV) model we here interrogated the entire apical DHBV ε (Dε) half for sequence- and structure-dependent determinants of in vitro priming activity, replication, and, in part, in vivo infectivity. This revealed single-strandedness of the bulge, a following G residue plus the loop subsequence GUUGU as the few key determinants for priming and initiation site selection; unexpectedly, they functioned independently of a specific structure context. These data provide new mechanistic insights into avihepadnaviral replication initiation, and they imply a new concept towards a feasible in vitro priming system for human HBV.
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