Sequence- and structure-specific determinants in the interaction between the RNA encapsidation signal and reverse transcriptase of avian hepatitis B viruses (original) (raw)
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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, 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.
Journal of Virology
Hepadnaviruses replicate by reverse transcription of an RNA intermediate within subviral core particles in the cytoplasm of infected hepatocytes. Recognition of the epsilon encapsidation signal located on the 5' end of the pregenomic RNA by the viral polymerase occurs early in core particle assembly. The epsilon sequences contain a set of nested inverted repeats which form a stable stem-loop structure shown to play a role in RNA packaging and recently implicated as the site of initiation of minus-strand DNA synthesis. We have introduced a variety of site-directed mutations into the epsilon sequences of human hepatitis B virus to study their effects on viral replication in transfected HuH7 cells. We have identified two classes of mutations: those which adversely affect packaging and those which package RNA but adversely affect DNA synthesis. Analysis of these mutants has allowed us to identify separate features of the epsilon cis-acting signal which function in the processes of R...
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.
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.
Nucleic Acids Research, 2004
Protein-primed replication of hepatitis B viruses (HBVs) is initiated by the chaperone dependent binding of the reverse transcriptase (P protein) to the bulged « stem-loop on the pregenomic RNA, and the «-templated synthesis of the 5 0 terminal nucleotides of the first DNA strand. How P protein recognizes the initiation site is poorly understood. In mammalian HBVs and in duck HBV (DHBV) the entire stem-loop is extensively base paired; in other avian HBVs the upper stem regions have a low base pairing potential. Initiation can be reconstituted with in vitro translated DHBV, but not HBV, P protein and DHBV « (D«) RNA. Employing the SELEX method on a constrained library of D« upper stem variants, we obtained a series of well-binding aptamers. Most contained C-rich consensus motifs with very low base pairing potential; some supported initiation, others did not. Consensus-based secondary mutants allowed to pin down this functional difference to the residues flanking the conserved loop, and an unpaired U. In vitro active consensus sequences also supported virus replication. Hence, most of the upper stem acts as a spacer, which, if not base paired, warrants accessibility of relevant anchor residues. This suggests that the base paired D« represents an exceptional rather than a prototypic avian HBV « signal, and it offers an explanation as to why attempts to in vitro reconstitute initiation with human HBV have thus far failed.
Distinct families of cis-acting RNA replication elements epsilon from hepatitis B viruses
RNA biology, 2012
The hepadnavirus encapsidation signal, epsilon (ε), is an RNA structure located at the 5' end of the viral pregenomic RNA. It is essential for viral replication and functions in polymerase protein binding and priming. This structure could also have potential regulatory roles in controlling the expression of viral replicative proteins. In addition to its structure, the primary sequence of this RNA element has crucial functional roles in the viral lifecycle. Although the ε elements in hepadnaviruses share common critical functions, there are some significant differences in mammalian and avian hepadnaviruses, which include both sequence and structural variations. Here we present several covariance models for ε elements from the Hepadnaviridae. The model building included experimentally determined data from previous studies using chemical probing and NMR analysis. These models have sufficient similarity to comprise a clan. The clan has in common a highly conserved overall structur...
Hepatology, 2002
Hepadnaviral replication requires the concerted action of the polymerase and core proteins to ensure selective packaging of the RNA pregenome into nucleocapsids. Virus assembly is initiated by cis-preferential binding of polymerase to the encapsidation signal ⑀, present on pregenomic RNA. Using the duck hepatitis B virus (DHBV) model, we analyzed how core protein is recruited to the RNA/polymerase preassembly complex. Two sets of transcomplementation assays were performed in cotransfected hepatoma cells. First, a replication-competent DHBV construct was tested for its ability to rescue replication of genomes bearing mutations within the core region. Self-packaging of wild-type pregenomes was more efficient than cross-packaging of core-deficient pregenomes, and this bias was strongly enhanced if mutant pregenomes coded for self-assembly-competent, but packaging-deficient, core proteins. Second, the site of wild-type core protein translation, i.e., pregenomic RNA (cis) or separate messenger RNA (trans), was analyzed for its effect on the phenotype of a previously described dominant-negative (DN) DHBV core protein mutant. This mutant forms chimeric nucleocapsids with wild-type core proteins and blocks reverse transcription within most, but not all, mixed particles. Strikingly, suppression of viral DNA synthesis by the mutant increased 100-fold when wild-type core protein was provided in trans. Our results suggest that recruitment of core protein to the DHBV preassembly complex occurs in a cis-preferential manner. This mechanism may account for the leakiness of DN DHBV core protein mutants targeting reverse transcription. (HEPATOLOGY 2002;35:209-216.)