Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming
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 elem...
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doaj-7431dee15fa947bb97373f7fe4a05b752020-12-08T01:57:44ZengNature Publishing GroupScientific Reports2045-23222017-08-017111610.1038/s41598-017-07657-zFew basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-primingMarkus Gajer0Katharina Dörnbrack1Christine Rösler2Bernadette Schmid3Jürgen Beck4Michael Nassal5University Hospital Freiburg, Department of Internal Medicine II/Molecular BiologyUniversity Hospital Freiburg, Department of Internal Medicine II/Molecular BiologyUniversity Hospital Freiburg, Department of Internal Medicine II/Molecular BiologyUniversity Hospital Freiburg, Department of Internal Medicine II/Molecular BiologyUniversity Hospital Freiburg, Department of Internal Medicine II/Molecular BiologyUniversity Hospital Freiburg, Department of Internal Medicine II/Molecular BiologyAbstract 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.https://doi.org/10.1038/s41598-017-07657-z |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Markus Gajer Katharina Dörnbrack Christine Rösler Bernadette Schmid Jürgen Beck Michael Nassal |
spellingShingle |
Markus Gajer Katharina Dörnbrack Christine Rösler Bernadette Schmid Jürgen Beck Michael Nassal Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming Scientific Reports |
author_facet |
Markus Gajer Katharina Dörnbrack Christine Rösler Bernadette Schmid Jürgen Beck Michael Nassal |
author_sort |
Markus Gajer |
title |
Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming |
title_short |
Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming |
title_full |
Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming |
title_fullStr |
Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming |
title_full_unstemmed |
Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming |
title_sort |
few basepairing-independent motifs in the apical half of the avian hbv ε rna stem-loop determine site-specific initiation of protein-priming |
publisher |
Nature Publishing Group |
series |
Scientific Reports |
issn |
2045-2322 |
publishDate |
2017-08-01 |
description |
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. |
url |
https://doi.org/10.1038/s41598-017-07657-z |
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