Abstract
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.
Highlights
Hepadnaviruses, with the important human pathogen hepatitis B virus (HBV) as their prototype, are small enveloped DNA viruses that replicate via protein-primed reverse transcription of a pregenomic RNA intermediate[1, 2] (Fig. 1A)
In the largely double-helical wild-type DHBV ε (Dε) the distance between bulge and loop is around 5–6 nm[26]
To modulate this distance with minimal induction of new, alternative basepairings we used as parent a Dε variant, S1230, in which the top part of the upper stem lacks basepairing potential; the five basepairs on top of the bulge and the two basepairs closing the tetraloop can form (Fig. 2A), as in heron HBV (HHBV) ε26
Summary
Hepadnaviruses, with the important human pathogen hepatitis B virus (HBV) as their prototype, are small enveloped DNA viruses that replicate via protein-primed reverse transcription of a pregenomic (pg) RNA intermediate[1, 2] (Fig. 1A). In vitro HBV RNP complex formation has been achieved but without detectable DNA synthesis activity[13]; we refer to this a non-productive binding. Priming activity of recombinant full-length or near full-length P protein requires cellular chaperones, as present in rabbit reticulocyte lysate[6, 17,18,19] or added in substance[18], in particular heat shock proteins Hsc[70] and Hsp[404, 5, 20] This chaperone dependence is overcome by terminal truncations in P protein[20, 21], with the respective “miniDP” proteins providing the most feasible systems to investigate Dε-dependent protein-priming. The actual presence of both elements in free HBV and DHBV ε RNA was biochemically confirmed[6, 23,24,25], and NMR investigations revealed further details, including non-canonical basepairs, in the previously assumed (“classical”) heptaand hexaloop sequences capping the upper stem, creating a tetraloop in Dε26 and a pseudotriloop in HBV ε27
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