SELEX-derived aptamers of the duck hepatitis B virus RNA encapsidation signal distinguish critical and non-critical residues for productive initiation of reverse transcription (original) (raw)
Related papers
Journal of Biological Chemistry, 1999
Hepatitis B viruses replicate through reverse transcription of an RNA intermediate. In contrast to retroviral reverse transcriptases, their replication enzyme, P protein, does not use a nucleic acid primer but initiates DNA synthesis de novo from within an RNA stem-loop structure called ⑀. A short DNA oligonucleotide is copied from ⑀ and covalently attached to P protein, and then synthesis is arrested. The information for initiation site selection and synthesis arrest must be contained in the structure of the P protein/⑀ complex. Because P protein activity depends on cellular chaperones this complex can as yet only be generated by in vitro translation of duck hepatitis B virus P protein in rabbit reticulocyte lysate; functional interaction with its cognate RNA element D⑀ can be monitored by the covalent labeling of P protein during primer synthesis. Combining this in vitro priming reaction and a set of chimeric RNA-DNA D⑀ analogues, we found that only five ribose residues in the 57-nucleotide stem-loop were sufficient to provide a functional template; these are a single residue in the template region and the two base pairs at the tip of the lower stem. The base identities in the very same region are essential as well. The presence of this 2-OH-and base-dependent determinant shortly downstream of the initiation site suggests a mechanism that can account for both initiation site selection and programmed primer synthesis arrest.
Journal of Virology, 2001
Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure () on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli, followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of -dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.
Journal of virology, 1997
Hepatitis B viruses (HBVs) replicate by reverse transcription of an RNA intermediate. Packaging of this RNA pregenome into nucleocapsids and replication initiation depend crucially on the interaction of the reverse transcriptase, P protein, with the cis-acting, 5' end-proximal encapsidation signal epsilon. The overall secondary structure is similar in all of the hepadnaviral epsilon signals, with a lower and an upper stem, separated by a bulge, and an apical loop. However, while epsilon is almost perfectly conserved in all mammalian viruses, the epsilon signals of duck HBV (DHBV) and heron HBV (D epsilon and H epsilon, respectively) differ substantially in their upper stem regions, both in primary sequence and in secondary structure; nonetheless, H epsilon interacts productively with DHBV P protein, as shown by its ability to stimulate priming, i.e., the covalent attachment of a deoxynucleoside monophosphate to the protein. In this study, we extensively mutated the variable and ...
PLoS ONE, 2013
Replication of hepatitis B virus (HBV) via protein-primed reverse transcription is initiated by binding of the viral P protein to the conserved e stem-loop on the pregenomic (pg) RNA. This triggers encapsidation of the complex and the e-templated synthesis of a short P protein-linked DNA oligonucleotide (priming) for subsequent minus-strand DNA extension. e consists of a lower and upper stem, a bulge containing the priming template, and an apical loop. The nonhelical subelements are considered important for DNA synthesis and pgRNA packaging whereas the role of the upper stem is not well characterized. Priming itself could until recently not be addressed because in vitro generated HBV P -e complexes showed no activity. Focussing on the four A residues at the base and tip of the upper e stem and the two U residues in the loop we first investigated the impact of 24 mutations on viral DNA accumulation in transfected cells. While surprisingly many mutations were tolerated, further analyzing the negatively acting mutations, including in a new cell-free priming system, revealed divergent position-related impacts on pgRNA packaging, priming activity and possibly initiation site selection. This genetic separability implies that the e RNA undergoes multiple distinct interactions with P protein as pgRNA encapsidation and replication initiation progress, and that the strict conservation of e in nature may reflect its optimal adaptation to comply with all of them. The data further define the most attractive mutants for future studies, including as decoys for interference with HBV replication.
Scientific reports, 2017
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 GUUG...
Molecular and cellular biology, 1998
The DNA genome of a hepatitis B virus is generated by reverse transcription of the RNA pregenome. Replication initiation does not involve a nucleic acid primer; instead, the hepadnavirus P protein binds to the structured RNA encapsidation signal epsilon, from which it copies a short DNA primer that becomes covalently linked to the enzyme. Using in vitro-translated duck hepatitis B virus (DHBV) P protein, we probed the secondary structure of the protein-bound DHBV epsilon RNA (Depsilon) and observed a marked conformational change compared to free Depsilon RNA. Several initiation-competent mutant RNAs with a different free-state structure were similarly altered, whereas a binding-competent but initiation-deficient variant was not, indicating the importance of the rearrangement for replication initiation and suggesting a mechanistic coupling to encapsidation.
Novel mechanism for reverse transcription in hepatitis B viruses
Journal of Virology, 1993
Reverse transcription of all retroviruses and most retroid elements requires tRNA as a primer for DNA synthesis. However, in hepatitis B viruses the viral polymerase itself acts as a primer for reverse transcription (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992). We have now demonstrated that in order to prime DNA synthesis, the polymerase binds to an RNA hairpin, which then serves as a template for the formation of a short DNA primer that is covalently linked to protein. Following its synthesis, the nascent DNA strand apparently dissociates from its template and reanneals with complementary sequences at the 3' end of the RNA genome, where DNA synthesis continues. Since this RNA hairpin also functions as a packaging signal for viral RNA, hepadnaviruses have adopted a replication strategy that relies on the same signal for two biochemically distinct events, RNA packaging and reverse transcription. This mechanism is without precedent among all known retroid elements and among o...
Virology, 1997
Hepatitis B viruses replicate via reverse transcription of an RNA intermediate. This RNA pregenome serves as mRNA and is packaged into capsids and reverse transcribed. Both processes require the interaction of the viral reverse transcriptase, P protein, with the 5-proximal e-signals on the pregenome. For e of human hepatitis B virus (HBV), the presence of a functionally important stem -loop structure with a central bulge, part of which acts as template for a short primer of first-strand DNA synthesis, has been experimentally confirmed. Based on phylogeny and its functional similarities to e, the De-signal of duck hepatitis B virus (DHBV) had been proposed to have a similar structure which does not, however, correspond to the most stable computer prediction. We have therefore experimentally determined the secondary structures of De and of the He-signal of heron hepatitis B virus which differs considerably from De in primary sequence yet interacts productively with DHBV P protein. Our data support an HBV e-like structure for both De and He; in particular the bulge is highly conserved, in accord with its special function in replication. However, the apical loop in He is much enlarged suggesting that, by an induced-fit mechanism, both RNAs may adopt a new, probably similar conformation in the complex with P protein. ᭧ 1997 Academic Press
PLoS ONE, 2011
Background: The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the e RNA stem-loop on pregenomic (pg) RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking e in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified.
Journal of Virology, 2012
Hepadnaviruses, including the pathogenic hepatitis B virus (HBV), replicate their small DNA genomes through protein-primed reverse transcription, mediated by the terminal protein (TP) domain in their P proteins and an RNA stem-loop, ⑀, on the pregenomic RNA (pgRNA). No direct structural data are available for P proteins, but their reverse transcriptase (RT) domains contain motifs that are conserved in all RTs (box A to box G), implying a similar architecture; however, experimental support for this notion is limited. Exploiting assays available for duck HBV (DHBV) but not the HBV P protein, we assessed the functional consequences of numerous mutations in box E, which forms the DNA primer grip in human immunodeficiency virus type 1 (HIV-1) RT. This substructure coordinates primer 3=-end positioning and RT subdomain movements during the polymerization cycle and is a prime target for nonnucleosidic RT inhibitors (NNRTIs) of HIV-1 RT. Box E was indeed critical for DHBV replication, with the mutations affecting the folding, ⑀ RNA interactions, and polymerase activity of the P protein in a position-and amino acid side chain-dependent fashion similar to that of HIV-1 RT. Structural similarity to HIV-1 RT was underlined by molecular modeling and was confirmed by the replication activity of chimeric P proteins carrying box E, or even box C to box E, from HIV-1 RT. Hence, box E in the DHBV P protein and likely the HBV P protein forms a primer grip-like structure that may provide a new target for anti-HBV NNRTIs.