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Dörnbrack K, Beck J, Nassal M. Relaxing the restricted structural dynamics in the human hepatitis B virus RNA encapsidation signal enables replication initiation in vitro. PLoS Pathog 2022; 18:e1010362. [PMID: 35259189 PMCID: PMC8903280 DOI: 10.1371/journal.ppat.1010362] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2021] [Accepted: 02/10/2022] [Indexed: 11/18/2022] Open
Abstract
Hepadnaviruses, including hepatitis B virus (HBV) as a major human pathogen, replicate their tiny 3 kb DNA genomes by capsid-internal protein-primed reverse transcription of a pregenomic (pg) RNA. Initiation requires productive binding of the viral polymerase, P protein, to a 5´ proximal bipartite stem-loop, the RNA encapsidation signal ε. Then a residue in the central ε bulge directs the covalent linkage of a complementary dNMP to a Tyr sidechain in P protein´s Terminal Protein (TP) domain. After elongation by two or three nucleotides (nt) the TP-linked DNA oligo is transferred to a 3´ proximal acceptor, enabling full-length minus-strand DNA synthesis. No direct structural data are available on hepadnaviral initiation complexes but their cell-free reconstitution with P protein and ε RNA (Dε) from duck HBV (DHBV) provided crucial mechanistic insights, including on a major conformational rearrangement in the apical Dε part. Analogous cell-free systems for human HBV led at most to P—ε binding but no detectable priming. Here we demonstrate that local relaxation of the highly basepaired ε upper stem, by mutation or via synthetic split RNAs, enables ε-dependent in vitro priming with full-length P protein from eukaryotic translation extract yet also, and without additional macromolecules, with truncated HBV miniP proteins expressed in bacteria. Using selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) we confirm that upper stem destabilization correlates with in vitro priming competence and show that the supposed bulge-closing basepairs are largely unpaired even in wild-type ε. We define the two 3´ proximal nt of this extended bulge as main initiation sites and provide evidence for a Dε-like opening of the apical ε part upon P protein binding. Beyond new HBV-specific basic aspects our novel in vitro priming systems should facilitate the development of high-throughput screens for priming inhibitors targeting this highly virus-specific process. Chronic hepatitis B virus (HBV) infection puts >250 million people at an increased risk for severe liver disease. Current treatments can control but rarely cure infection. HBV features a 3,200 bp DNA genome, generated by reverse transcription of a pregenomic (pg) RNA. To initiate DNA synthesis the viral polymerase, P protein, employs a stem-loop on pgRNA, ε, to covalently link a defined first nucleotide to its Terminal Protein (TP) domain. This protein-priming is highly virus-specific yet poorly understood. More is known for duck HBV (DHBV) where, different from HBV, protein-priming was successfully reconstituted in vitro years ago. One insight was that gaining priming-competence involves opening of the apical stem in DHBV ε RNA (Dε); in HBV ε the more extensive basepairing might restrict such dynamics. Here we relaxed these constraints by identifying functional but less stably folded, including split, HBV ε variants. Several such variants supported in vitro priming, including in a simple two-component-system employing a shortened recombinant P protein. Amongst other data the new cell-free systems yielded a first view on a major conformational change in HBV ε RNA bound to P protein, highlighting the importance of RNA dynamics for the human virus. Beyond furthering basic understanding our data should facilitate screening for protein-priming inhibitors as new anti-HBV agents.
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Affiliation(s)
- Katharina Dörnbrack
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Freiburg, Germany
| | - Jürgen Beck
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Freiburg, Germany
- * E-mail: (JB); , (MN)
| | - Michael Nassal
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Freiburg, Germany
- * E-mail: (JB); , (MN)
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Yao Y, Yang B, Cao H, Zhao K, Yuan Y, Chen Y, Zhang Z, Wang Y, Pei R, Chen J, Hu X, Zhou Y, Lu M, Wu C, Chen X. RBM24 stabilizes hepatitis B virus pregenomic RNA but inhibits core protein translation by targeting the terminal redundancy sequence. Emerg Microbes Infect 2018; 7:86. [PMID: 29760415 PMCID: PMC5951808 DOI: 10.1038/s41426-018-0091-4] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2018] [Revised: 03/22/2018] [Accepted: 04/03/2018] [Indexed: 02/07/2023]
Abstract
The terminal redundancy (TR) sequence of the 3.5-kb hepatitis B virus (HBV) RNA contains sites that govern many crucial functions in the viral life cycle, including polyadenylation, translation, RNA packaging, and DNA synthesis. In the present study, RNA-binding motif protein 24 (RBM24) is shown to be involved in the modulation of HBV replication by targeting the TR of HBV RNA. In HBV-transfected hepatoma cell lines, both knockdown and overexpression of RBM24 led to decreased HBV replication and transcription. Ectopic expression of RBM24 inhibited HBV replication, which was partly restored by knockdown of RBM24, indicating that a proper level of RBM24 was required for HBV replication. The regulation of RBM24 of HBV replication and translation was achieved by the interaction between the RNA-binding domains of RBM24 and both the 5' and 3' TR of 3.5-kb RNA. RBM24 interacted with the 5' TR of HBV pregenomic RNA (pgRNA) to block 80S ribosome assembly on HBV pgRNA and thus inhibited core protein translation, whereas the interaction between RBM24 and the 3' TR enhanced the stability of HBV RNA. Finally, the regulatory function of RBM24 on HBV replication was further confirmed in a HBV infection model. In conclusion, the present study demonstrates the dual functions of RBM24 by interacting with different TRs of viral RNA and reveals that RBM24 is an important host gene for HBV replication.
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Affiliation(s)
- Yongxuan Yao
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Bo Yang
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Huang Cao
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Kaitao Zhao
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Yifei Yuan
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Yingshan Chen
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Zhenhua Zhang
- Department of Infectious Diseases, The First Affiliated Hospital, Anhui Medical University, Hefei, 230022, China
- School of Pharmacy, Anhui Medical University, Hefei, 230022, China
| | - Yun Wang
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Rongjuan Pei
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Jizheng Chen
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Xue Hu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Yuan Zhou
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Mengji Lu
- Institute of Virology, University Hospital of Essen, Essen, Germany
| | - Chunchen Wu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
| | - Xinwen Chen
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
- University of Chinese Academy of Sciences, Beijing, China.
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3
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Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming. Sci Rep 2017; 7:7120. [PMID: 28769080 PMCID: PMC5541001 DOI: 10.1038/s41598-017-07657-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Accepted: 06/28/2017] [Indexed: 12/12/2022] Open
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.
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Bhoola NH, Kramvis A. Expression of wild-type or G1862T mutant HBe antigen of subgenotype A1 of hepatitis B virus and the unfolded protein response in Huh7 cells. J Gen Virol 2017; 98:1422-1433. [PMID: 28678685 DOI: 10.1099/jgv.0.000793] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
The G1862T mutation, which occurs most frequently in subgenotype A1 of the hepatitis B virus (HBV), results in a valine to phenylalanine substitution at the -3 position of the signal peptide cleavage site at the amino end of the precore/core (preC/C) precursor protein. The objective of this study was to functionally characterize the G1862T mutation relative to its wild-type counterpart in subgenotype A1. Huh7 cells were transfected with subgenotype A1 replication-competent plasmids, with and without G1862T. Secretion of HBsAg and HBeAg, preC/C/HBeAg expression in the secretory pathway, activation of the unfolded protein response (UPR) and subsequent activation of apoptosis were monitored. The introduction of G1862T did not affect HBsAg expression. Cells transfected with the G1862T subgenotype A1 plasmid showed decreased expression of intracellular HBcAg and of nuclear preC/C/HBeAg and extracellular HBeAg, when compared to cells transfected with its wild-type counterpart as a result of the accumulation of the mutant protein in the endoplasmic reticulum (ER) and ER-Golgi intermediate compartment (ERGIC) . This accumulation of preC/C/HBeAg protein in the ER led to the earlier activation of the three UPR pathways, but not to an increase in apoptosis. Therefore, it is evident that the presence of G1862T in subgenotype A1 does not completely abolish HBeAg expression, but affects the rate of HBeAg maturation, its passage through the secretory pathway and activation of the UPR. Increase in ER stress can result in liver damage, which has been shown to be a contributing factor to hepatocarcinogenesis and may explain why G1862T is frequently found in subgenotype A1 from liver disease patients.
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Affiliation(s)
- Nimisha Harshadrai Bhoola
- Hepatitis Virus Diversity Research Unit, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg, South Africa
| | - Anna Kramvis
- Hepatitis Virus Diversity Research Unit, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg, South Africa
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Castelain S, Descamps V, Brochot E, Helle F, Duverlie G, Nguyen-Khac E, François C. High association of T1858-G1896 precore mutations with impaired base pairing and high hepatitis B virus DNA levels in HBeAg-negative chronically infected patients. Arch Virol 2017; 162:1913-1920. [PMID: 28289975 DOI: 10.1007/s00705-017-3312-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2016] [Accepted: 02/21/2017] [Indexed: 12/20/2022]
Abstract
The progression of liver disease in hepatitis B virus (HBV) infection is fostered by active virus replication. Mutations in the basal core promoter (BCP) and precore (PC) regions of the HBV genome are known to have an impact on viral replication. The aim of the present study was to assess the correlation of mutation profiles in the BCP and PC regions with the viral load in HBeAg-negative chronically infected patients. The HBV genotype, BCP/PC mutations, serum HBV DNA levels, and associated serological markers were analyzed in 92 HBeAg-negative chronically infected patients. Sequence analysis of the BCP and PC regions revealed variability of 19% and 24.1%, respectively. This variability was primarily associated with five critical positions (1753, 1762, 1764, 1896 and 1899). An elevated HBV viral load (>20,000 IU/ml) was classically correlated with F2-F4 liver fibrosis, elevated serum alanine aminotransferase levels, 1762/1764 and 1753 combination mutations, and surprisingly, with an 1858T-1896G double mutation that impairs base pairing at the base of the bulge in the ε encapsidation signal. An analysis of covariance confirmed the independent nature of the relationship between the 1858T-1896G double mutation and the HBV viral load. In conclusion, independently of conventional parameters, this study demonstrates that a high serum HBV DNA level was also associated with PC 1858-1896 mutations. These BCP/PC mutations may have important clinical implications as predictive factors for HBV DNA increase.
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Affiliation(s)
- Sandrine Castelain
- Virology Department, Centre de Biologie Humaine, Centre Hospitalo-Universitaire Amiens Picardie, 80054, Amiens Cedex, France. .,EA4294, Université de Picardie Jules Verne, Amiens, France.
| | - Véronique Descamps
- Virology Department, Centre de Biologie Humaine, Centre Hospitalo-Universitaire Amiens Picardie, 80054, Amiens Cedex, France.,EA4294, Université de Picardie Jules Verne, Amiens, France
| | - Etienne Brochot
- Virology Department, Centre de Biologie Humaine, Centre Hospitalo-Universitaire Amiens Picardie, 80054, Amiens Cedex, France.,EA4294, Université de Picardie Jules Verne, Amiens, France
| | - François Helle
- EA4294, Université de Picardie Jules Verne, Amiens, France
| | - Gilles Duverlie
- Virology Department, Centre de Biologie Humaine, Centre Hospitalo-Universitaire Amiens Picardie, 80054, Amiens Cedex, France.,EA4294, Université de Picardie Jules Verne, Amiens, France
| | - Eric Nguyen-Khac
- Hepatology Department, Centre Hospitalo-Universitaire Amiens Picardie, Amiens, France
| | - Catherine François
- Virology Department, Centre de Biologie Humaine, Centre Hospitalo-Universitaire Amiens Picardie, 80054, Amiens Cedex, France.,EA4294, Université de Picardie Jules Verne, Amiens, France
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Feng H, Chen P, Zhao F, Nassal M, Hu K. Evidence for multiple distinct interactions between hepatitis B virus P protein and its cognate RNA encapsidation signal during initiation of reverse transcription. PLoS One 2013; 8:e72798. [PMID: 23977352 PMCID: PMC3748129 DOI: 10.1371/journal.pone.0072798] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2013] [Accepted: 07/11/2013] [Indexed: 12/27/2022] Open
Abstract
Replication of hepatitis B virus (HBV) via protein-primed reverse transcription is initiated by binding of the viral P protein to the conserved ε stem-loop on the pregenomic (pg) RNA. This triggers encapsidation of the complex and the ε-templated synthesis of a short P protein-linked DNA oligonucleotide (priming) for subsequent minus-strand DNA extension. ε 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 - ε complexes showed no activity. Focussing on the four A residues at the base and tip of the upper ε 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 ε RNA undergoes multiple distinct interactions with P protein as pgRNA encapsidation and replication initiation progress, and that the strict conservation of ε 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.
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Affiliation(s)
- Hui Feng
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Ping Chen
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Fei Zhao
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Michael Nassal
- University Hospital Freiburg, Department of Internal Medicine II/Molecular Biology, Freiburg, Germany
- * E-mail: (MN); (KH)
| | - Kanghong Hu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
- Biomedical Center, Hubei University of Technology, Wuhan, China
- * E-mail: (MN); (KH)
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7
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In vitro epsilon RNA-dependent protein priming activity of human hepatitis B virus polymerase. J Virol 2012; 86:5134-50. [PMID: 22379076 DOI: 10.1128/jvi.07137-11] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed ε (Hε) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. We have purified HP from human cells that retained Hε binding activity in vitro. Furthermore, HP purified as a complex with Hε, but not HP alone, displayed in vitro protein priming activity. While the HP-Hε interaction in vitro and in vivo required the Hε internal bulge, but not its apical loop, and was not significantly affected by the cap-Hε distance, protein priming required both the Hε apical loop and internal bulge, as well as a short distance between the cap and Hε, mirroring the requirements for RNA packaging. These studies have thus established new HBV protein priming and RNA binding assays that should greatly facilitate the dissection of the requirements and molecular mechanisms of HP-Hε interactions, RNA packaging, and protein priming.
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McCann MD, Lim GFS, Manni ML, Estes J, Klapec KA, Frattini GD, Knarr RJ, Gratton JL, Serra MJ. Non-nearest-neighbor dependence of the stability for RNA group II single-nucleotide bulge loops. RNA (NEW YORK, N.Y.) 2011; 17:108-19. [PMID: 21088109 PMCID: PMC3004052 DOI: 10.1261/rna.2306911] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/07/2010] [Accepted: 10/23/2010] [Indexed: 05/24/2023]
Abstract
Thirty-one RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each sequence were determined. The bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The data were used to develop a model to predict the free energy of an RNA duplex containing a single-nucleotide bulge. The destabilization of the duplex by the bulge was primarily related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. Since there is an ambiguity of the bulge position for group II bulges, several different stem combinations are possible. The destabilization of group II bulge loops is similar to the destabilization of group I bulge loops, if the second least stable stem is used to predict the influence of the group II bulge. In-line structure probing of the group II bulge loop embedded in a hairpin indicates that the bulged nucleotide is the one positioned farther from the hairpin loop.
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Affiliation(s)
- Michael D McCann
- Department of Chemistry, Allegheny College, Meadville, Pennsylvania 16335, USA
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Das AT, Berkhout B. HIV-1 evolution: frustrating therapies, but disclosing molecular mechanisms. Philos Trans R Soc Lond B Biol Sci 2010; 365:1965-73. [PMID: 20478891 PMCID: PMC2880118 DOI: 10.1098/rstb.2010.0072] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Replication of HIV-1 under selective pressure frequently results in the evolution of virus variants that replicate more efficiently under the applied conditions. For example, in patients on antiretroviral therapy, such evolution can result in variants that are resistant to the HIV-1 inhibitors, thus frustrating the therapy. On the other hand, virus evolution can help us to understand the molecular mechanisms that underlie HIV-1 replication. For example, evolution of a defective virus mutant can result in variants that overcome the introduced defect by restoration of the original sequence or by the introduction of additional mutations in the viral genome. Analysis of the evolution pathway can reveal the requirements of the element under study and help to understand its function. Analysis of the escape routes may generate new insight in the viral life cycle and result in the identification of unexpected biological mechanisms. We have developed in vitro HIV-1 evolution into a systematic research tool that allows the study of different aspects of the viral replication cycle. We will briefly review this method of forced virus evolution and provide several examples that illustrate the power of this approach.
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Affiliation(s)
| | - Ben Berkhout
- Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
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10
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Inoue J, Ueno Y, Nagasaki F, Wakui Y, Kondo Y, Fukushima K, Niitsuma H, Shimosegawa T. Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis. Virology 2009; 395:202-9. [PMID: 19850315 DOI: 10.1016/j.virol.2009.09.028] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2009] [Revised: 06/22/2009] [Accepted: 09/23/2009] [Indexed: 12/17/2022]
Abstract
A plasmid carrying 1.3-fold HBV genome was constructed from a HBV strain that caused five consecutive cases of fulminant hepatitis (pBFH2), and HepG2 cells were transfected with pBFH2 or its variants. The pBFH2 construct with A1762T/G1764A, G1862T, and G1896A showed the largest amount of core particle-associated intracellular HBV DNA, but no significant increase of extracellular HBV DNA in comparison with the wild construct, suggesting that these mutations might work together for retention of the replicative intermediates in the cells. The retention might relate to the localization of hepatitis B core antigen (HBcAg) in the nucleus of HepG2, which was observed by confocal fluorescence microscopy. HBcAg immunohistochemical examination of liver tissue samples obtained from the consecutive fulminant hepatitis patients showed stronger staining in the nucleus than acute hepatitis patients. In conclusion, the fulminant HBV strain caused retention of the core particles and the core particle-associated HBV DNA in the cells.
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Affiliation(s)
- Jun Inoue
- Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aoba, Sendai 980-8574, Japan.
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11
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Abstract
Mutational analysis of the viral genome is frequently used to study the role of sequence or structural elements in HIV-1 replication. Many laboratories that use this approach have occasionally come across revertant viruses that overcome an introduced defect either by restoration of the original sequence or by the introduction of additional mutations in the viral genome. Similarly, replication of a wild type virus under selective pressure, due to the presence of inhibitors or due to specific culture settings, may result in the appearance of evolved variants that replicate more efficiently under the applied conditions. We have developed in vitro HIV-1 evolution from an anecdotal event to a systematic research tool to study different aspects of the viral replication cycle. In this manuscript, we will briefly review the method of forced virus evolution to study HIV-1 biology and provide several examples that illustrate the power of this method, as it frequently yielded interesting and unexpected information about the mechanism of virus replication.
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Affiliation(s)
- Ben Berkhout
- Laboratory of Experimental Virology, Academic Medical Center, Amsterdam, The Netherlands
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12
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Hu J, Lin L. RNA-protein interactions in hepadnavirus reverse transcription. Front Biosci (Landmark Ed) 2009; 14:1606-18. [PMID: 19273150 DOI: 10.2741/3328] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
The small DNA genome of hepadnaviruses is replicated by reverse transcription via an RNA intermediate. This RNA "pregenome" contains important signals that control critical steps of viral replication, including RNA packaging, initiation of reverse transcription, and elongation of minus strand DNA, through specific interactions with the viral reverse transcriptase, the capsid protein, and host factors. In particular, the interaction between the viral reverse transcriptase and RNA pregenome requires a host chaperone complex composed of the heat shock protein 90 and its cochaperones.
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Affiliation(s)
- Jianming Hu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
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13
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Nassal M. Hepatitis B viruses: reverse transcription a different way. Virus Res 2008; 134:235-49. [PMID: 18339439 DOI: 10.1016/j.virusres.2007.12.024] [Citation(s) in RCA: 282] [Impact Index Per Article: 16.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2007] [Revised: 11/28/2007] [Accepted: 12/05/2007] [Indexed: 02/07/2023]
Abstract
Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is the type member of the Hepadnaviridae, hepatotropic DNA viruses that replicate via reverse transcription. Beyond long-established differences to retroviruses in gene expression and overall replication strategy newer work has uncovered additional distinctions in the mechanism of reverse transcription per se. These include protein-priming by the unique extra terminal protein domain of the reverse transcriptase (RT) utilizing an RNA hairpin for de novo initiation of first strand DNA synthesis, and the strict dependence of this process on cellular chaperones. Recent in vitro reconstitution systems enabled first biochemical insights into this multifactorial reaction, complemented by high resolution structural information on the RNA, though not yet the protein, level. Genetic approaches have revealed long-distance interactions in the nucleic acid templates as an important factor enabling the puzzling template switches required to produce the relaxed circular (RC) DNA found in infectious virions. Finally, the failure of even potent HBV RT inhibitors to eliminate nuclear covalently closed circular (ccc) DNA, the functional equivalent of integrated proviral DNA, has spurred a renewed interest in the mechanism of cccDNA generation. These new developments are in the focus of this review.
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Affiliation(s)
- Michael Nassal
- University Hospital Freiburg, Internal Medicine 2/Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany.
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14
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Abstract
Aptamers are rare nucleic acid ligands, which can be concocted in the laboratory from the randomized pool of molecules by affinity and amplification processes. Aptamers have several properties as they can be applied complementarily to antibodies and have several advantages over antibodies. In the past, several aptamers have been selected with a view to develop antiviral agents for therapeutic applications. This review summarizes potent antiviral aptamers and their strategies to prevent the viral replication.
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Affiliation(s)
- S C B Gopinath
- Functional Nucleic Acids Group, Institute for Biological Resources and Functions and Center for Applied Near Field Optics Research, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan.
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15
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Chandra PK, Banerjee A, Datta S, Chakravarty R. G1862T mutation among hepatitis B virus-infected individuals: association with viral genotypes and disease outcome in Kolkata, Eastern India. Intervirology 2007; 50:173-80. [PMID: 17259736 DOI: 10.1159/000098960] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2006] [Accepted: 07/27/2006] [Indexed: 12/28/2022] Open
Abstract
OBJECTIVE To study the prevalence of G1862T mutation in hepatitis B virus (HBV) isolates among Eastern Indian patients and its relationship with genotypes, HBeAg status and disease manifestation. METHODS HBV DNA was isolated from patients, amplified by nested PCR and sequenced directly. RESULTS Of the 102 patients, 32 were HBeAg positive and 70 HBeAg negative; 55, 24 and 23 isolates were infected with genotypes D, A and C, respectively. G1862T was detected in 18 samples, 15 (83%) of them belonged to genotype A (subgenotype HBV/A1), 3 (17%) to genotype D. This mutation was more frequent in HBeAg-negative than in HBeAg-positive patients (21 vs. 9%), whereas in HBV/A1 it was as common in HBeAg-positive as in HBeAg-negative patients and significantly associated with T1762/A1764 mutation. The mean viral load was lower in patients with G1862T mutation. Furthermore, this mutation was common in various clinical outcomes. CONCLUSION In our community, G1862T mutation was predominantly found in HBV/A1 isolates irrespective of HBeAg status. Moreover this mutation could not be correlated to the clinical outcome. These findings indicate that the G1862T mutation is probably a part of the natural variability of HBV/A1.
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16
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Abstract
Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, ε, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV), now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cell-free systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the ε RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.
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MESH Headings
- Animals
- Base Sequence
- Capsid/physiology
- DNA, Circular/genetics
- DNA, Circular/physiology
- DNA, Viral/genetics
- DNA, Viral/physiology
- Disease Models, Animal
- Ducks
- Hepatitis B Virus, Duck/genetics
- Hepatitis B Virus, Duck/physiology
- Hepatitis B virus/genetics
- Hepatitis B virus/physiology
- Humans
- Molecular Sequence Data
- RNA/genetics
- RNA/physiology
- RNA, Circular
- RNA, Viral/genetics
- RNA, Viral/physiology
- RNA-Directed DNA Polymerase/physiology
- Virus Replication/genetics
- Virus Replication/physiology
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Affiliation(s)
- Juergen Beck
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Hugstetter Street 55, D-79106 Freiburg, Germany
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17
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Gopinath SCB. Methods developed for SELEX. Anal Bioanal Chem 2006; 387:171-82. [PMID: 17072603 DOI: 10.1007/s00216-006-0826-2] [Citation(s) in RCA: 248] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2006] [Revised: 08/29/2006] [Accepted: 09/01/2006] [Indexed: 02/07/2023]
Abstract
SELEX (systematic evolution of ligands by exponential enrichment) is a process that involves the progressive purification from a combinatorial library of nucleic acid ligands with a high affinity for a particular target by repeated rounds of partitioning and amplification. With the development of aptamer technology over the last decade, various modified SELEX processes have arisen that allow various aptamers to be developed against a wide variety of molecules, irrespective of the target size. In the present review, the separation methods used in such SELEX processes are reviewed.
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Affiliation(s)
- Subash Chandra Bose Gopinath
- Functional Nucleic Acids Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
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Hu J, Boyer M. Hepatitis B virus reverse transcriptase and epsilon RNA sequences required for specific interaction in vitro. J Virol 2006; 80:2141-50. [PMID: 16474122 PMCID: PMC1395402 DOI: 10.1128/jvi.80.5.2141-2150.2006] [Citation(s) in RCA: 78] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA by the viral reverse transcriptase (RT). Using an in vitro reconstitution system whereby the cellular heat shock protein 90 chaperone system activates recombinant HBV RT for specific epsilon binding, we have defined the protein and RNA sequences required for specific HBV RT-epsilon interaction in vitro. Our results indicated that approximately 150 amino acid residues from the terminal protein domain and 230 from the RT domain were necessary and sufficient for epsilon binding. With respect to the epsilon RNA sequence, its internal bulge and, in particular, the first nucleotide (C) of the bulge were specifically required for RT binding. Sequences from the upper portion of the lower stem and the lower portion of the upper stem also contributed to RT binding, as did the base pairing of the upper portion and the single unpaired U residue of the upper stem. Surprisingly, the apical loop of epsilon, known to be required for RNA packaging, was entirely dispensable for RT binding. A comparison of the requirements for in vitro RT-epsilon interaction with those for in vivo pregenomic RNA (pgRNA) packaging clearly indicated that RT-epsilon interaction was necessary but not sufficient for pgRNA packaging. In addition, our results suggest that recognition of some epsilon sequences by the RT may be required specifically for viral DNA synthesis.
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Affiliation(s)
- Jianming Hu
- Department of Microbiology and Immunology, H107, The Penn State University College of Medicine, Hershey, 17033, USA.
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19
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Sugauchi F, Kumada H, Acharya SA, Shrestha SM, Gamutan MTA, Khan M, Gish RG, Tanaka Y, Kato T, Orito E, Ueda R, Miyakawa Y, Mizokami M. Epidemiological and sequence differences between two subtypes (Ae and Aa) of hepatitis B virus genotype A. J Gen Virol 2004; 85:811-820. [PMID: 15039524 DOI: 10.1099/vir.0.79811-0] [Citation(s) in RCA: 116] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Complete nucleotide sequences of 19 hepatitis B virus (HBV) isolates of genotype A (HBV/A) were determined and analysed along with those of 20 previously reported HBV/A isolates. Of the 19 HBV/A isolates, six including three from Japan and three from the USA clustered with the 14 HBV/A isolates from Western countries. The remaining 13 isolates including four from The Philippines, two from India, three from Nepal and four from Bangladesh clustered with the six HBV/A isolates reported from The Philippines, South Africa and Malawi. Due to distinct epidemiological distributions, genotype A in the 20 HBV isolates was classified into subtype Ae (e for Europe), and that in the other 19 into subtype Aa (a for Asia and Africa) provisionally. The 19 HBV/Aa isolates had a sequence variation significantly greater than that of the 20 HBV/Ae isolates (2.5+/-0.3 % vs 1.1+/-0.6 %, P<0.0001); they differed by 5.0+/-0.4 % (4.1-6.4 %). The double mutation (T1762/A1764) in the core promoter was significantly more frequent in HBV/Aa isolates than in HBV/Ae isolates (11/19 or 58 % vs 5/20 or 25 %, P<0.01). In the pregenome encapsidation (epsilon) signal, a point mutation from G to A or T at nt 1862 was detected in 16 of the 19 (84 %) HBV/Aa isolates but not in any of the 20 HBV/Ae isolates, which may affect virus replication and translation of hepatitis B e antigen. Subtypes Aa and Ae of genotype A deserve evaluation for any clinical differences between them, with a special reference to hepatocellular carcinoma prevalent in Africa.
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Affiliation(s)
- Fuminaka Sugauchi
- Departments of Internal Medicine and Molecular Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
- Departments of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Hiromitsu Kumada
- Department of Gastroenterology, Toranomon Hospital, Tokyo, Japan
| | - Subrat A Acharya
- Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India
| | | | | | - Mobin Khan
- Department of Hepatology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh
| | - Robert G Gish
- Hepatology and Gastroenterology, California Pacific Medical Center, San Francisco, USA
| | - Yasuhito Tanaka
- Departments of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Takanobu Kato
- Departments of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Etsuro Orito
- Departments of Internal Medicine and Molecular Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Ryuzo Ueda
- Departments of Internal Medicine and Molecular Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | | | - Masashi Mizokami
- Departments of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
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20
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You S, Stump DD, Branch AD, Rice CM. A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication. J Virol 2004; 78:1352-66. [PMID: 14722290 PMCID: PMC321395 DOI: 10.1128/jvi.78.3.1352-1366.2004] [Citation(s) in RCA: 174] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5' and 3' ends of the RNA genome should provide new insights into HCV RNA replication.
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Affiliation(s)
- Shihyun You
- Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10021, USA
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21
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Flodell S, Cromsigt J, Schleucher J, Kidd-Ljunggren K, Wijmenga S. Structure elucidation of the hepatitis B virus encapsidation signal by NMR on selectively labeled RNAs. J Biomol Struct Dyn 2002; 19:627-36. [PMID: 11843624 DOI: 10.1080/07391102.2002.10506769] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Abstract
Hepatitis B virus (HBV) HBV is DNA virus with a unique replication strategy, which involves reverse transcription of its pregenomic RNA. Essential for this reverse transcription are the 5'- and 3'-ends of its pregenomic RNA (5'-RT-RNA and 3'-RT-RNA, respectively) which form conserved bulged stem-loop structures. The 5'-RT-RNA consists of a 67 nucleotide bulged stem-loop structure, epsilon, which constitutes the signal for encapsidation of the pregenomic RNA and subsequent reverse transcription. The reverse transcriptase (RT) initially binds to the completely conserved apical loop of epsilon and a 4-nucleotide primer is synthesized from the adjacent 6-nucleotide bulge. Structural studies of epsilon can provide important parameters required for the design of RNA targeted anti- viral drugs directed against Hepatitis B virus. NMR studies of large RNA systems (> ca. 50 nucleotides) require novel approaches, e.g., different labeling schemes and reduction of the system into separate structural building blocks. Recently, a new method of synthesizing (13)C/(15)N/(2)H labeled nucleotides has been developed based on converting specifically labeled glucose and bases into nucleotides by using enzymes from the pentose phosphate pathway and nucleotide and salvage pathways. These NTPs give a large freedom in designing different labeling patterns in in vitro synthesized RNAs under study for NMR. This opens up the way for NMR studies of RNAs that are considerably above the present size limit (up to 150 nucleotides). Here this new technique is applied for structural studies on 27, 36 and 61 nucleotides long RNA fragments, mimicking different regions of epsilon.
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Affiliation(s)
- Sara Flodell
- Department of Medical Biosciences, Medical Biophysics, Umeå University, S-901 87 Umeå, Sweden
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22
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Schaaf SG, Beck J, Nassal M. A small 2'-OH- and base-dependent recognition element downstream of the initiation site in the RNA encapsidation signal is essential for hepatitis B virus replication initiation. J Biol Chem 1999; 274:37787-94. [PMID: 10608840 DOI: 10.1074/jbc.274.53.37787] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
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 epsilon. A short DNA oligonucleotide is copied from epsilon 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/epsilon 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 Depsilon 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 Depsilon 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.
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Affiliation(s)
- S G Schaaf
- Department of Internal Medicine II, University Hospital Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany
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23
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Abstract
The hepatitis B virus (HBV) and other members of the hepadnaviridae replicate by reverse transcription of an RNA intermediate, pregenomic RNA (pgRNA). pgRNA is also translated into core protein and polymerase (reverse transcriptase) protein. Before being reverse transcribed, pgRNA is sequestrated from the cytoplasm by being packaged, together with polymerase, into subviral particles composed of core protein. For pgRNA to be encapsidated, its 5' end is folded into a stem-loop structure, known as the encapsidation signal or epsilon (epsilon). This stable bipartite stem-loop structure contains a bulge and an apical loop. Besides encapsidation, epsilon is involved in the activation of polymerase, in template restriction and in the initiation of DNA synthesis by reverse transcription. HBV DNA encoding epsilon forms part of the template that is translated into the precore/core fusion protein that is in turn post-translationally modified to produce hepatitis B e antigen (HBeAg). The DNA encoding epsilon may be recombinogenic. Mutations within epsilon can affect its function and sequence conservation within epsilon in natural isolates is therefore high. epsilon could provide a practical target for antiviral therapy.
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Affiliation(s)
- A Kramvis
- Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa
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24
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Kramvis A, Kew MC, Bukofzer S. Hepatitis B virus precore mutants in serum and liver of Southern African Blacks with hepatocellular carcinoma. J Hepatol 1998; 28:132-41. [PMID: 9537850 DOI: 10.1016/s0168-8278(98)80212-2] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND/AIM The aim of this study was to sequence the precore region of HBV isolated from serum and tumorous and non-tumorous liver tissue from patients with hepatocellular carcinoma to identify mutations that might play a role in malignant transformation. METHODS HBV DNA was extracted from 62 sera, 14 tumorous and 12 non-tumorous liver tissue samples of patients with hepatocellular carcinoma, amplified by the polymerase chain reaction and sequenced directly. RESULTS Thirty-nine patients were HBeAg-negative and 23 HBeAg-positive. Missense mutations were present predominantly in HBeAg-negative sera. The most common missense mutation, a guanine to thymine transversion, occurred at nucleotide 1862 in the bulge of the encapsidation signal; it was more prevalent in HBeAg-negative (10/39) than in HBeAg-positive patients (1/23) (p = 0.03). Mutations known to prevent HBeAg synthesis were detected in seven sera; five with an 1896 stop-codon mutation, one with an 1817 nonsense mutation, and one with a frameshift mutation caused by an insertion between 1838 and 1839. Missense mutations and deletions were present more often in tumorous tissue derived from HBsAg-negative patients. In the tumours missense mutations occurred at position 1862 and 1899, and the deletions affected direct repeat 1 and/or the encapsidation signal and included the x gene stop-codon. CONCLUSIONS The 1862 mutation, and other missense mutations and deletions detected in the precore gene, may disrupt HBV DNA replication and/or signal peptide cleavage leading to HBeAg-negativity. Disruption of viral replication may promote integration of unencapsidated replicative intermediates and hence contribute to hepatocarcinogenesis.
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Affiliation(s)
- A Kramvis
- Medical Research Council, Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa
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25
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Jiang H, Loeb DD. Insertions within epsilon affect synthesis of minus-strand DNA before the template switch for duck hepatitis B virus. J Virol 1997; 71:5345-54. [PMID: 9188604 PMCID: PMC191772 DOI: 10.1128/jvi.71.7.5345-5354.1997] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Duck hepatitis B virus (DHBV) is a DNA virus that replicates via reverse transcription of a pregenomic RNA (pgRNA). Synthesis of the first strand of DNA (minus-strand DNA) for DHBV can be divided into two steps: (i) synthesis of the first four nucleotides of minus-strand DNA, which is primed by the viral polymerase (P) protein and copied from the sequence 5'-UUAC-3' within the phylogenetically conserved bulge in the encapsidation signal (epsilon) near the 5' end of pgRNA; and (ii) a template switch of the four-nucleotide minus-strand DNA from epsilon to an acceptor site near the 3' end of pgRNA and synthesis of a complete minus-strand DNA. To understand why only four nucleotides of minus-strand DNA were synthesized before the template switch, we introduced small insertions immediately 5' to the UUAC sequence in epsilon and determined whether these epsilon variants were competent for protein priming and whether minus strands longer than four nucleotides were synthesized. Then we determined, in cell culture, whether the longer minus-strand DNAs were competent to undergo a template switch. Also, we analyzed the structure of the epsilon variants, in solution. We found that the epsilon variants were functional for protein priming and RNA encapsidation and that the insertions were copied into minus-strand DNA. However, two mutant viruses that contained two different three-nucleotide insertions failed to synthesize minus-strand DNA efficiently from the acceptor site, even though seven nucleotides of the donor and acceptor sites were identical. These results suggest that the length and/or sequence of the minus-strand DNA copied from epsilon can be important for an efficient template switch. The RNA structural analysis of the epsilon variants indicated alteration in the position and size of the bulge. Overall, these results are consistent with the notion that the template within epsilon is limited to four nucleotides because the remaining two nucleotides located within the bulge are inaccessible for polymerization.
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Affiliation(s)
- H Jiang
- McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706, USA
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26
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Osborne SE, Ellington AD. Nucleic Acid Selection and the Challenge of Combinatorial Chemistry. Chem Rev 1997; 97:349-370. [PMID: 11848874 DOI: 10.1021/cr960009c] [Citation(s) in RCA: 389] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Affiliation(s)
- Scott E. Osborne
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405
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27
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Berkhout B, Klaver B, Das AT. Forced evolution of a regulatory RNA helix in the HIV-1 genome. Nucleic Acids Res 1997; 25:940-7. [PMID: 9023102 PMCID: PMC146548 DOI: 10.1093/nar/25.5.940] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and simian immunodeficiency viruses suggests that the thermodynamic stability of the helix is fine-tuned. We demonstrated previously that mutant HIV-1 genomes with a stabilized or destabilized hairpin are severely replication-impaired. In this study, we found that the mutant with a destabilized polyA hairpin structure is conditionally defective. Whereas reduced replication is measured in infections at the regular temperature (37 degrees C), this mutant is more fit than the wild-type virus at reduced temperature (33 degrees C). This observation of a temperature-dependent replication defect underscores that the stability of this RNA structure is critical for function. An extensive analysis of revertant viruses was performed to further improve the understanding of the critical sequence and structural features of the element under scrutiny. The virus mutants with a stabilized or destabilized hairpin were used as a starting point in multiple, independent selections for revertant viruses with compensatory mutations. Both mutants reverted to hairpins with wild-type stability along various pathways by acquisition of compensatory mutations. We identified 19 different revertant HIV-1 forms with improved replication characteristics, providing a first look at some of the peaks in the total sequence landscape that are compatible with virus replication. These experiments also highlight some general principles of RNA structure building.
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Affiliation(s)
- B Berkhout
- Academic Medical Center, University of Amsterdam, Department of Human Retrovirology, PO Box 22700, 1100 DE Amsterdam, The Netherlands.
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28
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29
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Abstract
Hepatitis B virus (HBV), the causative agent of type B hepatitis in humans, is the prototypic member of the hepadnaviridae, a family of small enveloped DNA-containing viruses with pronounced host and tissue specificity. This property has greatly hampered progress in understanding the initial events of infection, i.e. attachment, penetration and uncoating. After the discovery, originally made with the duck hepatitis B virus (DHBV), that hepadnaviruses replicate by reverse transcription, DNA transfection of cloned wild-type and mutant HBV genomes into cell lines supporting virion formation has revealed the molecular mechanisms of the late steps of the infectious cycle in some detail. During the last few years, such studies have emphasized the differences between hepadnaviral and retroviral replication. Very recent research, however, indicates that the border separating the two viral families may not be as strict as previously thought. In this article, we will briefly summarize the pertinent differences, and will then focus on the new data, with particular emphasis on the initiation of reverse transcription.
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Affiliation(s)
- M Nassal
- Zentrum für Molekulare Biologie (ZMBH), University of Heidelberg, Germany
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30
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Abstract
In vitro selection experiments are now routinely used to identify functional nucleic acid residues and structures, and have become a tool for studying molecular recognition, molecular biology, and molecular evolution. Technical innovations that have been made during the past year include the use of modified monomers to increase stability and photocross-linking reagents to improve affinity. These advances should dramatically increase the utility of aptamers in the future.
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Affiliation(s)
- K W Uphoff
- Department of Chemistry, Indiana University, Bloomington 47405, USA
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31
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Rieger A, Nassal M. Specific hepatitis B virus minus-strand DNA synthesis requires only the 5' encapsidation signal and the 3'-proximal direct repeat DR1. J Virol 1996; 70:585-9. [PMID: 8523575 PMCID: PMC189849 DOI: 10.1128/jvi.70.1.585-589.1996] [Citation(s) in RCA: 56] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Human hepatitis B virus (HBV) is a small DNA virus that replicates inside the viral nucleocapsid by reverse transcription of an RNA intermediate, the pregenome. The sequences encompassing the encapsidation signal epsilon and the direct repeat DR1 are present in two copies of this terminally redundant transcript. We have recently shown that HBV minus-strand DNA synthesis involves transfer of a short DNA primer copied from 5'-epsilon to 3'-DR1 (DR1*). Using transfection of HBV genomes with lesions in 3'-epsilon, and 5'-DR1 and its preceding sequence, we tested whether these additional elements contribute to the specificity of the transfer reaction. However, while some mutations affected proper plus-strand DNA formation, 5'-epsilon and DR1* were completely sufficient for correct minus-strand DNA production.
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Affiliation(s)
- A Rieger
- Zentrum für Molekulare Biologie, Universität Heidelberg, Germany
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32
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Affiliation(s)
- M Nassal
- Center for Molecular Biology, University of Heidelberg, Germany
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33
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Beck J, Nassal M. Efficient hammerhead ribozyme-mediated cleavage of the structured hepatitis B virus encapsidation signal in vitro and in cell extracts, but not in intact cells. Nucleic Acids Res 1995; 23:4954-62. [PMID: 8559651 PMCID: PMC307499 DOI: 10.1093/nar/23.24.4954] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Hepatitis B virus (HBV), the causative agent of B-type hepatitis in man, is a small enveloped DNA virus that replicates through reverse transcription of an RNA intermediate, the terminally redundant RNA pregenome. An essential highly conserved cis-element present twice on this RNA is the encapsidation signal epsilon, a stem-loop structure that is critical for pregenome packaging and reverse transcription. Epsilon is hence an attractive target for antiviral therapy. Its structure, however, is a potential obstacle to antivirals whose action depends on hybridization, e.g. ribozymes. Here we demonstrate effective in vitro cleavage inside epsilon by hammerhead ribozymes containing flanking sequences complementary to an adjacent less structured region. Upon co-transfection with a HBV expression construct corresponding ribozymes embedded in a U6 snRNA context led to a significant, though modest, reduction in the steady-state level of HBV pregenomes. Inactive ribozyme mutants revealed that antisense effects contributed substantially to this reduction, however, efficient epsilon cleavage by the intracellularly expressed ribozymes was observed in Mg(2+)-supplemented cell lysates. Artificial HBV pregenomes carrying the ribozymes in cis and model RNAs lacking all HBV sequences except epsilon exhibited essentially the same behaviour. Hence, neither the absence of co-localization of ribozyme and target nor a viral component, but rather a cellular factor(s), is responsible for the strikingly different ribozyme activities inside cells and in cellular extracts.
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Affiliation(s)
- J Beck
- Zentrum für Molekulare Biologie, Universität Heidelberg, Germany
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