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Olenginski LT, Attionu SK, Henninger EN, LeBlanc RM, Longhini AP, Dayie TK. Hepatitis B Virus Epsilon (ε) RNA Element: Dynamic Regulator of Viral Replication and Attractive Therapeutic Target. Viruses 2023; 15:1913. [PMID: 37766319 PMCID: PMC10534774 DOI: 10.3390/v15091913] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2023] [Revised: 09/01/2023] [Accepted: 09/05/2023] [Indexed: 09/29/2023] Open
Abstract
Hepatitis B virus (HBV) chronically infects millions of people worldwide, which underscores the importance of discovering and designing novel anti-HBV therapeutics to complement current treatment strategies. An underexploited but attractive therapeutic target is ε, a cis-acting regulatory stem-loop RNA situated within the HBV pregenomic RNA (pgRNA). The binding of ε to the viral polymerase protein (P) is pivotal, as it triggers the packaging of pgRNA and P, as well as the reverse transcription of the viral genome. Consequently, small molecules capable of disrupting this interaction hold the potential to inhibit the early stages of HBV replication. The rational design of such ligands necessitates high-resolution structural information for the ε-P complex or its individual components. While these data are currently unavailable for P, our recent structural elucidation of ε through solution nuclear magnetic resonance spectroscopy marks a significant advancement in this area. In this review, we provide a brief overview of HBV replication and some of the therapeutic strategies to combat chronic HBV infection. These descriptions are intended to contextualize our recent experimental efforts to characterize ε and identify ε-targeting ligands, with the ultimate goal of developing novel anti-HBV therapeutics.
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Affiliation(s)
- Lukasz T. Olenginski
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
- Department of Biochemistry, University of Colorado, Boulder, CO 80309, USA
| | - Solomon K. Attionu
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
| | - Erica N. Henninger
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
| | - Regan M. LeBlanc
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
| | - Andrew P. Longhini
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
- Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
| | - Theodore K. Dayie
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
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Rajput MK. Mutations and methods of analysis of mutations in Hepatitis B virus. AIMS Microbiol 2020; 6:401-421. [PMID: 33364535 PMCID: PMC7755589 DOI: 10.3934/microbiol.2020024] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2020] [Accepted: 10/23/2020] [Indexed: 12/13/2022] Open
Abstract
Immunization programmes against hepatitis-B are being carried out since more than three decades but still HBV is a major public health problem. Hepatitis B virus (HBV) genome consists of circular and partial double stranded DNA. Due to partial double stranded DNA, it uses an RNA intermediate during replication. This replicative strategy of HBV and lack of polymerase proofreading activity give rise to error occurrences comparable to retroviruses. The low fidelity of polymerase, overlapping reading frames and high replication rate produces many non-identical variants at every cycle of replication. Therefore, HBV spreads with mutations and variations. The mutations have been reported both in non-structural as well as structural genes of HBV genome. Recent advances in molecular biology have made easier to analyse these mutations. Hepatitis B antiviral therapy and immunization are all influenced by genetic variability. The analysis and understanding of these mutations are important for therapy against hepatitis B and updating of diagnostic tools. The present review discusses about mutations occurring in whole HBV genome. The mutation occurring both in structural and non-structural genes and non-coding regions have been described in details. It is much more informative because most of literature available, covers only individual gene or DNA regions of HBV.
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Kheirabad AK, Farshidfar G, Nasrollaheian S, Gouklani H. Prevalence and Characteristics of Precore Mutation in Iran and Its Correlation with Genotypes of Hepatitis B. Electron Physician 2017; 9:4114-4123. [PMID: 28607644 PMCID: PMC5459281 DOI: 10.19082/4114] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2016] [Accepted: 11/03/2016] [Indexed: 01/05/2023] Open
Abstract
Introduction Mutation of the HBV precore gene prevents the production of HBeAg, which is an important target for immune responses. Distribution of this mutation varies along with frequency of HBV genotypes in accordance with geographic and ethnic variations. The general objective of this study was to evaluate the prevalence and characteristics of precore mutation in Iran and its correlation with genotypes of hepatitis B. Methods In this cross-sectional study, viral DNA of 182 Iranian hepatitis B surface antigen positive patients who were admitted to Bandar Abbas Blood Transfusion Organization in 2012 and 2013 was retrieved from their serum samples. HBeAg, anti-HBe, and anti-HBc IgM diagnostic tests were performed using ELISA kits. Precore and Pre-S regions were amplified using specific primers and PCR thereafter to determine the genotypes; precore mutation, PCR, and restriction fragment length polymorphism (RFLP) methods also were applied. SPSS version 12 was used for data analysis by Mann–Whitney U test, Fisher’s exact probability test, and t-test. Results A total of 62 patients (34.1%) had precore mutation (A1896G), and genotype D was the predominant genotype in these patients, which was followed by an unknown genotype that was suspected for genotype B. Interestingly, the relationships between precore mutation and HBeAg (p=0.037) and genotype D (p=0.005) were significant; however, no correlation was observed between this mutation and acute or chronic hepatitis and sex of patients. Conclusion This study found high prevalence of precore mutations in southern Iran, which was significantly associated with HBeAg and genotype D.
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Affiliation(s)
- Ali Kargar Kheirabad
- Ph.D., Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
| | - Gholamreza Farshidfar
- Ph.D., Department of Biochemistry, School of Medicine, Hormozgan University of Medical Sciences, Iran
| | | | - Hamed Gouklani
- Ph.D., Molecular Medicine Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
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Baxa DM, Thekdi AD, Golembieski A, Krishnan PV, Sharif O, Kizy A, Shetron-Rama L, Jovanovich J, Chappell BJ, Snow-Lampart A, Borroto-Esoda K, Gordon SC. Evaluation of anti-HBV drug resistant mutations among patients with acute symptomatic hepatitis B in the United States. J Hepatol 2013; 58:212-6. [PMID: 23022497 DOI: 10.1016/j.jhep.2012.09.014] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2011] [Revised: 08/25/2012] [Accepted: 09/19/2012] [Indexed: 12/29/2022]
Abstract
BACKGROUND & AIMS Reported HBV drug resistance mutations among previously untreated patients with chronic hepatitis B are variable. Whether resistant HBV strains are transmitted in the acute setting is uncertain. We sought to document the presence of antiviral resistance (AVR) mutations in patients with acute HBV (AHB) infection. METHODS AHB infection was defined by HBsAg/IgM anti-HBc positivity, ALT>10X ULN and compatible clinical history. The TRUGENE HBV kit was used to perform genotyping and direct sequencing of the viral polymerase. INNO-LiPA HBV DRv2 and DRv3 were used to detect AVR mutations. Clonal sequencing was conducted on selected specimens. RESULTS Twenty-three patients were evaluated (mean age, 43 years; 54% male; 39% African American, 39% Caucasian, 13% Hispanic and 4% Asian). The mean peak ALT was 1554.2IU/L and mean peak total serum bilirubin was 12 mg/dl. The HBV DNA median viral load (N = 15) was 5.14 log(10)IU/ml. Nineteen patients were genotype A, and 1 each were genotype C, D, E and G. HBV drug resistance mutations were not detected by direct sequencing or INNO-LiPA. Clonal sequencing was conducted on 192 clones isolated from three patients and showed rtA181T, rtM250V and rtS202G mutations at an overall frequency of 1.54%, 1.39%, and 1.67% respectively. CONCLUSIONS We detected adefovir/lamivudine and entecavir relevant mutations in a minor population (<2%) of viral clones by clonal sequencing only. The clinical significance of these mutations is uncertain and may represent small populations of quasi-species vs. transmission of drug resistant strains.
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Affiliation(s)
- Dwayne M Baxa
- Infectious Disease, Henry Ford Hospital, Detroit, MI, USA.
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Zhu Y, Curtis M, Borroto-Esoda K. HBV DNA replication mediated by cloned patient HBV reverse transcriptase genes from HBV genotypes A–H and its use in antiviral phenotyping assays. J Virol Methods 2011; 173:340-6. [DOI: 10.1016/j.jviromet.2011.03.006] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2010] [Revised: 03/03/2011] [Accepted: 03/03/2011] [Indexed: 01/07/2023]
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Yokosuka O, Kurosaki M, Imazeki F, Arase Y, Tanaka Y, Chayama K, Tanaka E, Kumada H, Izumi N, Mizokami M, Kudo M. Management of hepatitis B: Consensus of the Japan Society of Hepatology 2009. Hepatol Res 2011; 41:1-21. [PMID: 21070536 DOI: 10.1111/j.1872-034x.2010.00739.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Recently, much progress has been made in the field of hepatitis B, such as natural history of the disease in relation to the amount of hepatitis B virus (HBV) DNA, genotypes of HBV influencing the natural course and treatment effects, mutations of HBV influencing the severity of the disease and development of hepatocellular carcinoma, and antiviral treatment such as nucleos(t)ide analogues and pegylated interferon. To make the consensus for the diagnosis, management and treatment of hepatitis B, a meeting was held during 45th annual meeting of Japan Society of Hepatology (JSH) in June 2009. In the meeting, recommendations and informative statements were discussed on the following subjects: (i) natural history of HBV infection; (ii) clinical implication of HBV genotypes; (iii) HBV mutations and their potential impact on pathogenesis of HBV infection; (iv) indications for antiviral treatment of chronic hepatitis B; (v) nucleos(t)ide analogues for chronic hepatitis B; and (vi) interferon therapy for chronic hepatitis B. The presenters reviewed the data on these subjects and proposed the consensus statements and recommendations. These statements were discussed among the organizers and presenters, and were approved by the participants of the meeting. In the current report, the relevant data were reviewed and the 12 consensus statements and nine recommendations on chronic hepatitis B were described.
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Affiliation(s)
- Osamu Yokosuka
- Department of Medicine and Clinical Oncology, Postgraduate School of Medicine, Chiba University, Japan
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Porterfield JZ, Dhason MS, Loeb DD, Nassal M, Stray SJ, Zlotnick A. Full-length hepatitis B virus core protein packages viral and heterologous RNA with similarly high levels of cooperativity. J Virol 2010; 84:7174-84. [PMID: 20427522 PMCID: PMC2898219 DOI: 10.1128/jvi.00586-10] [Citation(s) in RCA: 124] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2010] [Accepted: 04/22/2010] [Indexed: 01/01/2023] Open
Abstract
A critical feature of a viral life cycle is the ability to selectively package the viral genome. In vivo, phosphorylated hepatitis B virus (HBV) core protein specifically encapsidates a complex of pregenomic RNA (pgRNA) and viral polymerase; it has been suggested that packaging is specific for the complex. Here, we test the hypothesis that core protein has intrinsic specificity for pgRNA, independent of the polymerase. For these studies, we also evaluated the effect of core protein phosphorylation on assembly and RNA binding, using phosphorylated core protein and a phosphorylation mimic in which S155, S162, and S170 were mutated to glutamic acid. We have developed an in vitro system where capsids are disassembled and assembly-active core protein dimer is purified. With this protein, we have reassembled empty capsids and RNA-filled capsids. We found that core protein dimer bound and encapsidated both the HBV pregenomic RNA and heterologous RNA with high levels of cooperativity, irrespective of phosphorylation. In direct competition assays, no specificity for pregenomic RNA was observed. This suggests that another factor, such as the viral polymerase, is required for specific packaging. These results also beg the question of what prevents HBV core protein from assembling on nonviral RNA, preserving the protein for virus production.
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Affiliation(s)
- J. Zachary Porterfield
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, University Hospital Freiburg, Internal Medicine 2, Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany, Department of Microbiology, University of Mississippi, Jackson, Mississippi 39216
| | - Mary Savari Dhason
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, University Hospital Freiburg, Internal Medicine 2, Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany, Department of Microbiology, University of Mississippi, Jackson, Mississippi 39216
| | - Daniel D. Loeb
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, University Hospital Freiburg, Internal Medicine 2, Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany, Department of Microbiology, University of Mississippi, Jackson, Mississippi 39216
| | - Michael Nassal
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, University Hospital Freiburg, Internal Medicine 2, Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany, Department of Microbiology, University of Mississippi, Jackson, Mississippi 39216
| | - Stephen J. Stray
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, University Hospital Freiburg, Internal Medicine 2, Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany, Department of Microbiology, University of Mississippi, Jackson, Mississippi 39216
| | - Adam Zlotnick
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, University Hospital Freiburg, Internal Medicine 2, Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany, Department of Microbiology, University of Mississippi, Jackson, Mississippi 39216
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Sharma S, Sharma B, Singla B, Chawla YK, Chakraborti A, Saini N, Duseja A, Das A, Dhiman RK. Clinical significance of genotypes and precore/basal core promoter mutations in HBV related chronic liver disease patients in North India. Dig Dis Sci 2010; 55:794-802. [PMID: 20043209 DOI: 10.1007/s10620-009-1083-y] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/01/2009] [Accepted: 12/01/2009] [Indexed: 12/18/2022]
Abstract
PURPOSE Data on genotypes, basal core promoter (BCP) and precore mutants of hepatitis B virus and their association with different HBV related liver disease have been studied inadequately and are controversial. Thus, the aim of this study was to determine the incidence of BCP and precore HBV mutants and their relationship with HBV genotype and different stages of HBV related liver disease in North Indian patients. METHODS A total 273 patients with different stages of HBV related liver diseases were enrolled. Nested polymerase chain reaction (PCR) was used to amplify the BCP/PC regions. RFLP and direct sequencing were performed to validate the mutations identified in these regions. HBV genotyping was accomplished by multiplex PCR. RESULTS Genotype D was the predominant genotype found in each of the various HBV related liver diseases. The BCP mutation was found significantly more often in inactive carriers and compensated cirrhosis as compared to the other groups. The BCP mutation was present in 29.1% of patients with genotype D versus 17.1% with genotype A (P = 0.001). The precore mutation was also more frequently observed with genotype D compared with genotype A (36.9 vs. 4.8%, P = 0.0007). CONCLUSION Genotype D is predominant in North Indian patients. The BCP and precore mutations occur in one-third of HBV positive patients in association with the genotype D. We did not find any correlation with severity of liver disease with genotypes and mutations.
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Affiliation(s)
- Sanjeev Sharma
- Department of Hepatology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
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9
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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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10
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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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De Mitri MS, Morsica G, Cassini R, Bagaglio S, Andreone P, Bianchi G, Loggi E, Bernardi M. Low replication and variability of HBV pre-core in concomitant infection with hepatitis B and hepatitis C viruses. Arch Virol 2006; 152:395-404. [PMID: 16953307 DOI: 10.1007/s00705-006-0836-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2006] [Accepted: 07/11/2006] [Indexed: 12/18/2022]
Abstract
In an attempt to define the virological profile of HBV in HCV co-infection, we analysed the viral load, the infecting genotype, and the mutational pattern of the HBV pre-core region (pre-C), which is involved in viral encapsidation and DNA replication. Eighty-six patients were studied: 32 with serological HBV/HCV-1b co-infection (group BC), 32 infected by HBV alone (group B), and 22 by HCV-1b alone (group C). Sequence analysis of the HBV pre-S and pre-C regions identified genotypes and mutational patterns. The HBV viral load was significantly lower in group BC than in group B (p < 0.001), and the distribution of HBV pre-C mutations showed a higher prevalence of wild type in concomitant infection than in the control group (p < 0.006). The predominant HBV infecting strain was genotype D in both the BC (96%) and B (87%) groups. No difference was observed in HCV viremia levels between the two groups, whereas in HBV/HCV infection, the low levels of circulating HBV were closely associated with the low degree of variability of pre-C domain (p = 0.005). In conclusion, in HBV/HCV infection, the virological pattern was characterised by the dominance of HCV associated with lower HBV replication capacity and decreased emergence of HBV pre-C variants.
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Affiliation(s)
- M S De Mitri
- Department of Internal Medicine, Cardioangiology and Hepatology, University of Bologna, Bologna, Italy.
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12
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Flodell S, Petersen M, Girard F, Zdunek J, Kidd-Ljunggren K, Schleucher J, Wijmenga S. Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal. Nucleic Acids Res 2006; 34:4449-57. [PMID: 16945960 PMCID: PMC1636360 DOI: 10.1093/nar/gkl582] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5′ end of the RNA pregenome. Epsilon contains an apical stem–loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem–loop based on NOE, RDC and 1H chemical shift NMR data. The 1H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5′-CUGUGC-3′ folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.
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Affiliation(s)
| | - Michael Petersen
- Biophysical Chemistry, University of Nijmegen, Toernooiveld 16225ED Nijmegen, The Netherlands
- Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark5230 Odense M, Denmark
| | - Frederic Girard
- Biophysical Chemistry, University of Nijmegen, Toernooiveld 16225ED Nijmegen, The Netherlands
| | | | | | | | - Sybren Wijmenga
- Biophysical Chemistry, University of Nijmegen, Toernooiveld 16225ED Nijmegen, The Netherlands
- To whom correspondence should be addressed. Tel: +31 24 3653384/2678; Fax: +31 24 3652112;
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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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14
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Kramvis A, Kew MC. Relationship of genotypes of hepatitis B virus to mutations, disease progression and response to antiviral therapy. J Viral Hepat 2005; 12:456-64. [PMID: 16108759 DOI: 10.1111/j.1365-2893.2005.00624.x] [Citation(s) in RCA: 143] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Phylogenetic analysis has led to the classification of hepatitis B virus into eight genotypes, designated A to H. The genotypes have differences in biological properties and show heterogeneity in their global distribution. These attributes of the genotypes may account not only for differences in the prevalence of hepatitis B virus mutants in various geographic regions, but also be responsible for differences in the clinical outcome and response to antiviral treatment in different population groups.
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Affiliation(s)
- A Kramvis
- MRC/University Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa.
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15
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Karayiannis P, Carman WF, Thomas HC. Molecular Variations in the Core Promoter, Precore and Core Regions of Hepatitis B Virus, and their Clinical Significance. VIRAL HEPATITIS 2005:242-262. [DOI: 10.1002/9780470987131.ch15] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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França PHC, González JE, Munné MS, Brandão LH, Gouvea VS, Sablon E, Vanderborght BOM. Strong association between genotype F and hepatitis B virus (HBV) e antigen-negative variants among HBV-infected argentinean blood donors. J Clin Microbiol 2005; 42:5015-21. [PMID: 15528689 PMCID: PMC525202 DOI: 10.1128/jcm.42.11.5015-5021.2004] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
A number of reports have indicated an increased risk of cirrhosis and hepatocellular carcinoma in hepatitis B virus (HBV)-infected individuals carrying HBV e antigen (HBeAg)-negative variants. Although distinct core promoter and precore mutations distributed according to geographical locality and viral genotype have been reported, epidemiological data from South America are still scarce. The prevalences of HBV genotypes and core promoter and precore polymorphisms in 75 HBeAg-negative Argentinean blood donors were surveyed. The observed frequencies of HBV genotypes were 64.0% for genotype F, 17.3% each for genotypes A and D, and 1.3% for genotype C. Genotype F strains were widely distributed and significantly more prevalent in the northern region of the country (P < 0.001). An overall high proportion of a stop codon mutation (UAG) at precore codon 28 (66.7%) was observed. Wild-type codon 28 (UGG) was present in 29.3% of the samples, and the remaining 4.0% of samples had mixed variants. The combination of A at nucleotide (nt) 1762 and G at nt 1764 of the core promoter was found in 58.7% of the samples. The variant profiles--T at nt 1762 and A at nt 1764 or A at nt 1762 and A at nt 1764--were detected in 28.0 and 1.3% of the samples, respectively. The observed core promoter polymorphisms could not be related to the ratio of HBeAg to anti-HBeAg antibody, HBV genotype, or precore codon 28 status. Nevertheless, a clear association of genotype F and a precore stop codon mutation was found (P < 0.05). In conclusion, HBV genotype F and mutant codon 28 strains predominated and were strongly associated in a geographically broad Argentinean blood donor population.
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Affiliation(s)
- Paulo H C França
- Departmento de Virologia, Instituto de Microbiologia, Rio de Janeiro, Brazil
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17
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Flodell S, Schleucher J, Cromsigt J, Ippel H, Kidd-Ljunggren K, Wijmenga S. The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri-loop with two underlying pyrimidine bulges. Nucleic Acids Res 2002; 30:4803-11. [PMID: 12409471 PMCID: PMC135823 DOI: 10.1093/nar/gkf603] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2002] [Revised: 05/02/2002] [Accepted: 09/14/2002] [Indexed: 01/05/2023] Open
Abstract
Reverse transcription of hepatitis B virus (HBV) pregenomic RNA is essential for virus replication. In the first step of this process, HBV reverse transcriptase binds to the highly conserved encapsidation signal, epsilon (epsilon), situated near the 5' end of the pregenome. epsilon has been predicted to form a bulged stem-loop with the apical stem capped by a hexa- loop. After the initial binding to this apical stem- loop, the reverse transcriptase synthesizes a 4 nt primer using the bulge as a template. Here we present mutational and structural data from NMR on the apical stem-loop of epsilon. Application of new isotope-labeling techniques (13C/15N/2H-U-labeling) allowed resolution of many resonance overlaps and an extensive structural data set could be derived. The NMR data show that, instead of the predicted hexa-loop, the apical stem is capped by a stable UGU tri-loop closed by a C-G base pair, followed by a bulged out C. The apical stem contains therefore two unpaired pyrimidines (C1882 and U1889), rather than one as was predicted, spaced by 6 nt. C1882, the 3' neighbour to the G of the loop-closing C-G base pair, is completely bulged out, while U1889 is at least partially intercalated into the stem. Analysis of 205 of our own HBV sequences and 1026 strains from the literature, covering all genotypes, reveals a high degree of conservation of epsilon. In particular, the residues essential for this fold are either totally conserved or show rare non-disruptive mutations. These data strongly indicate that this fold is essential for recognition by the reverse transcriptase.
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Affiliation(s)
- Sara Flodell
- Department of Medical Biochemistry and Biophysics, Umeå University, S-901 87 Umeå, Sweden
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18
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Abstract
Chronic hepatitis B virus (HBV) infection is a major global cause of hepatocellular carcinoma (HCC). Individuals who are chronic carriers have a greater than 100-fold increased relative risk of developing the tumour. Several mechanisms of HBV-induced HCC have been proposed. Integration of HBV DNA into the genome of hepatocytes occurs commonly, although integration at cellular sites that are important for regulation of hepatocyte proliferation appears to be a rare event. Functions of the HBx protein are also potentially oncogenic. These include transcriptional activation of cellular growth regulatory genes, modulation of apoptosis and inhibition of nucleotide excision repair of damaged cellular DNA. The effects of HBx are mediated by interaction with cellular proteins and activation of cell signalling pathways. Variations in HBV genome sequences may be important in hepatocarcinogenesis, although their significance has not yet been completely elucidated. Necroinflammatory hepatic disease, which often accompanies chronic HBV infection, may contribute indirectly to hepatocyte transformation in a number of ways, including by facilitating HBV DNA integration, predisposing to the acquisition of cellular mutations and generating mutagenic oxygen reactive species. Although HCC is a malignancy with a poor prognosis, the availability of an effective vaccine against HBV infection, and its inclusion in the Expanded Programme of Immunization of many countries, augurs well for the eventual elimination of HBV-associated HCC.
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Affiliation(s)
- P Arbuthnot
- Department of Molecular Medicine and Haematology and Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand Medical School, 7 York Road, Parktown 2193, South Africa
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19
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François G, Kew M, Van Damme P, Mphahlele MJ, Meheus A. Mutant hepatitis B viruses: a matter of academic interest only or a problem with far-reaching implications? Vaccine 2001; 19:3799-815. [PMID: 11427251 DOI: 10.1016/s0264-410x(01)00108-6] [Citation(s) in RCA: 100] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Affiliation(s)
- G François
- WHO Collaborating Centre for Prevention and Control of Viral Hepatitis, Department of Epidemiology and Social Medicine, Universiteit Antwerpen, Universiteitsplein 1, B-2610 Antwerpen, Belgium.
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20
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Abstract
Chronic hepatitis B virus (HBV) infection is a major global cause of hepatocellular carcinoma (HCC). Individuals who are chronic carriers have a greater than 100-fold increased relative risk of developing the tumour. Several mechanisms of HBV-induced HCC have been proposed. Integration of HBV DNA into the genome of hepatocytes occurs commonly, although integration at cellular sites that are important for regulation of hepatocyte proliferation appears to be a rare event. Functions of the HBx protein are also potentially oncogenic. These include transcriptional activation of cellular growth regulatory genes, modulation of apoptosis and inhibition of nucleotide excision repair of damaged cellular DNA. The effects of HBx are mediated by interaction with cellular proteins and activation of cell signalling pathways. Variations in HBV genome sequences may be important in hepatocarcinogenesis, although their significance has not yet been completely elucidated. Necroinflammatory hepatic disease, which often accompanies chronic HBV infection, may contribute indirectly to hepatocyte transformation in a number of ways, including by facilitating HBV DNA integration, predisposing to the acquisition of cellular mutations and generating mutagenic oxygen reactive species. Although HCC is a malignancy with a poor prognosis, the availability of an effective vaccine against HBV infection, and its inclusion in the Expanded Programme of Immunization of many countries, augurs well for the eventual elimination of HBV-associated HCC.
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Affiliation(s)
- Patrick Arbuthnot
- Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School7 York Road, Parktown 2193, South Africa
- Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand Medical School7 York Road, Parktown 2193, South Africa
| | - Michael Kew
- Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand Medical School7 York Road, Parktown 2193, South Africa
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21
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Grandjacques C, Pradat P, Stuyver L, Chevallier M, Chevallier P, Pichoud C, Maisonnas M, Trépo C, Zoulim F. Rapid detection of genotypes and mutations in the pre-core promoter and the pre-core region of hepatitis B virus genome: correlation with viral persistence and disease severity. J Hepatol 2000; 33:430-9. [PMID: 11019999 DOI: 10.1016/s0168-8278(00)80279-2] [Citation(s) in RCA: 160] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
BACKGROUND/AIMS We aimed to clarify the clinical relevance of hepatitis B virus pre-core mutant detection in patients with chronic hepatitis B using a newly developed assay. METHODS Viral genotypes and pre-core mutations were studied in relation to viral persistence and liver disease severity using INNO-LiPA methodology. The study group included 151 patients with chronic hepatitis B, 85 positive for HBeAg (group I) and 66 positive for anti-HBe (group II). RESULTS The prevalence of viral genotypes in group I was: 64% A, 1% B, 15% C, 19% D, 0% E, 0% F and in group II: 39% A, 0% B, 2% C, 56% D, 2% E, 2% F (p<0.001). The prevalence of mutations at pre-core codon 28 (M2) was lower in group I (5%) than in group II (64%) (p<0.001). The prevalence of pre-core promoter mutations was also lower in group I (21%) than in group II (61%) (p<0.001). M2 mutations were more frequently detected in genotype D than in genotype A (p<0.001), while the other mutations were not influenced by viral genotype. Serum HBV DNA levels were significantly lower in group II versus group I (p<0.001), and in patients with any of the pre-core mutations versus wild-type sequence (p<0.01). Although cirrhosis was more frequent in group II (37%) versus group I (22%) and in patients with either one of the pre-core mutation (31%) versus wild-type sequence (25%), there was no statistical difference in liver severity assessed by ALT levels and Knodell score. CONCLUSION Pre-core mutants, whose molecular pattern is strongly dependent on viral genotypes, are associated with viral persistence in anti-HBe positive patients with ongoing chronic hepatitis B. The availability of this rapid assay should allow a precise monitoring of viral pre-core mutants during the course of chronic hepatitis B.
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22
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Abstract
The existence of HBV as quasispecies is thought to be favoured by the infidelity of HBV RT, which would account for the emergence of the many natural mutants with point substitutions. RT infidelity may also underlie the hypermutation phenomenon. Indeed, the oft-reported point mutation in the preC gene that leads to failure of HBeAg synthesis may be driven by a hypermutation-related mechanism. The presence of mutants with deletions and insertions involving single nucleotides and oligonucleotides at specific positions in the genome, and of mutants with deletions of even longer stretches particularly in the C gene, suggests that other mutagenic mechanisms operate. Candidates include slippage during mispairing between template and progeny DNA strand, the action of cellular topoisomerase I, and gene splicing using alternative donor and acceptor sites. Natural substitutions, deletions or insertions involving the Cp/ENII locus in the X gene can significantly alter the extent of viral replicative activity. Similar mutations occurring at other locations of Cp/ENII, and at B-cell epitope sites of the S gene are associated with failure to detect serological markers of HBV infection. HBV variation can also arise from recombination between coinfecting strains. S gene mutations that become evident following HBIG administration and HBV vaccination are all point substitutions, as are mutations in functional RT domains of the P gene after treatment with viral RT-inhibitory drugs. Widespread and long-term use of prophylactic and therapeutic agents may potentially generate serologically occult HBV variants that might become difficult to eradicate.
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Affiliation(s)
- S L Ngui
- Section of Hepatology, Rush-Presbyterian-St. Luke's Medical Centre, 1725 West Harrison, Chicago, Illinois 60612, USA
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23
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Günther S, Fischer L, Pult I, Sterneck M, Will H. Naturally occurring variants of hepatitis B virus. Adv Virus Res 1999; 52:25-137. [PMID: 10384235 DOI: 10.1016/s0065-3527(08)60298-5] [Citation(s) in RCA: 189] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- S Günther
- Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Federal Republic of Germany.
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24
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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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25
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Ghany MG, Ayola B, Villamil FG, Gish RG, Rojter S, Vierling JM, Lok AS. Hepatitis B virus S mutants in liver transplant recipients who were reinfected despite hepatitis B immune globulin prophylaxis. Hepatology 1998; 27:213-22. [PMID: 9425940 DOI: 10.1002/hep.510270133] [Citation(s) in RCA: 271] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Long-term hepatitis B immune globulin (HBIG) has been shown to reduce hepatitis B virus (HBV) reinfection in patients transplanted for hepatitis B. The aim of this study was to determine the prevalence of HBV S gene mutations in liver transplant recipients who developed recurrent hepatitis B despite HBIG prophylaxis, and to determine if these mutations can revert after withdrawal of HBIG. The entire S gene sequences in pre- and posttransplant sera from 20 patients who developed recurrent hepatitis B despite HBIG prophylaxis were compared. Ten (50%) patients had 18 amino acid substitutions involving the 'a' determinant in the posttransplant samples. These mutations were absent in 93% of the pretransplantation clones analyzed. There was a significant correlation between the development of mutations in the 'a' determinant region and the duration of HBIG therapy. Most of the mutations result in changes in predicted antigenicity of the S protein. During follow-up, mutations in 14 (78%) of 18 affected codons in the 'a' determinant region reverted back to the pretransplantation sequences; only 1 codon had a de novo change after the withdrawal of HBIG. Two control patients who did not receive HBIG had no change in the 'a' determinant in their posttransplantation samples. These data support the hypothesis that mutations in the S gene were induced or selected by immune pressure exerted by HBIG. HBV S mutants may play a role in HBV reinfection in liver transplant recipients who received HBIG prophylaxis.
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Affiliation(s)
- M G Ghany
- University of Michigan and VA Medical Center, Ann Arbor 48109, USA
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26
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Beck J, Nassal M. Sequence- and structure-specific determinants in the interaction between the RNA encapsidation signal and reverse transcriptase of avian hepatitis B viruses. J Virol 1997; 71:4971-80. [PMID: 9188560 PMCID: PMC191728 DOI: 10.1128/jvi.71.7.4971-4980.1997] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
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 the conserved positions in the upper stem of D epsilon and correlated the functional activities of the variant RNAs in a priming assay with secondary structure and physical P protein binding. These data revealed a proper overall structure, with the bulge and certain key residues, e.g., in the loop, being important constraints in protein binding. Many mutations at the evolutionarily variable positions complied with these criteria and yielded priming-competent RNAs. However, most mutants at the conserved positions outside the loop were defective in priming even though they had epsilon-like structures and bound to P protein; conversely, one point mutant in the loop with an apical structure different from those of D epsilon and H epsilon was priming competent. These results suggest that P protein binding can induce differently structured epsilon RNAs to adopt a new, common conformation, and they support an induced-fit model of the epsilon-P interaction in which both components undergo extensive structural alterations during formation of a priming-competent ribonucleoprotein complex.
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Affiliation(s)
- J Beck
- Zentrum für Molekulare Biologie, University of Heidelberg, Germany
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27
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Arauz-Ruiz P, Norder H, Visoná KA, Magnius LO. Genotype F prevails in HBV infected patients of Hispanic origin in Central America and may carry the precore stop mutant. J Med Virol 1997. [DOI: 10.1002/(sici)1096-9071(199704)51:4<305::aid-jmv8>3.0.co;2-9] [Citation(s) in RCA: 94] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
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28
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Zhang X, Zoulim F, Habersetzer F, Xiong S, Trépo C. Analysis of hepatitis B virus genotypes and pre-core region variability during interferon treatment of HBe antigen negative chronic hepatitis B. J Med Virol 1996. [PMID: 8825704 DOI: 10.1002/(sici)1096-9071(199601)48:1%3c8::aid-jmv2%3e3.0.co;2-e] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
The clinical importance of hepatitis B virus (HBV) genome variability has been reported recently. One example is the occurrence of hepatitis B virus pre-core mutants, which arise during spontaneous or interferon-induced seroconversion from HBeAg to anti-HBe and are thought to be selected by immune pressure. A survey of HBV pre-core mutants and viral genotypes in 35 HBeAg negative patients during interferon therapy was carried out to understand viral pathogenesis in this form of chronic hepatitis B. Seventeen patients responded to interferon therapy as assessed by the sustained normalization of serum ALT levels and the significant decrease of viremia levels. The response rate to interferon was independent of both initial serum viral DNA level and interferon doses. During interferon therapy, a significant decrease of M0 (wild-type pre-core sequence at pos. 1887-1908), M1 (TGG to TAG at pos. 1896) or M2 (TGG to TAG at pos. 1896, and GGC to GAC at pos. 1899) positive viral genomes was found in 48%, 42%, and 33% of patients, respectively. A higher response rate to interferon therapy was observed in patients infected with HBV genotype A (70%) or M0 positive strains (75%) as compared to patients infected with genotype D/E (40%) or M1/M2 positive strains (44%). The data support the hypothesis that pre-core defective HBV represent viral mutants with an increased capacity to resist exogenous alpha interferon. These findings emphasize that characterization of HBV genome variability prior to interferon therapy may help to predict antiviral response in HBeAg negative patients.
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29
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Kidd AH, Kidd-Ljunggren K. A revised secondary structure model for the 3'-end of hepatitis B virus pregenomic RNA. Nucleic Acids Res 1996; 24:3295-301. [PMID: 8811080 PMCID: PMC146111 DOI: 10.1093/nar/24.17.3295] [Citation(s) in RCA: 46] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
The polymerase encoded by human hepatitis B virus, which has reverse transcriptase and RNase H activity, binds to its pregenomic RNA template in a two-step process involving a terminal redundancy. Both first strand and second strand DNA synthesis involve primer translocation and second strand synthesis involves a template jump. Three parts of the genome, including the so-called core promoter, are known to show deletions in strains usually arising after long-standing HBV infection, but also in some patients treated with interferon. A computer-based study of RNA template folding in the core promoter region, accommodating well-known point mutations, has generated a model for the 3' DR1 primer binding site as being part of a superstructure encompassing an already well-established stem-loop. Depending on the identity of nucleotides 1762 and 1764, the DR1 region may assume two alternative secondary structures which stabilize it as a primer binding site to different extents. Remarkably, one of these structures includes a pronounced loop which coincides with at least 12 related deletions seen in HBV DNA from different patients. Thus according to the model, the 5'- and 3'-ends of pregenomic RNA, which share primary sequences but have separate functions, are not structural equivalents. An RNA superstructure near the 3'-end of all HBV transcripts could have far-reaching implications for the modulation of both genome replication and post-transcriptional processing.
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Affiliation(s)
- A H Kidd
- Department of Virology, University of Umeå, Sweden
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30
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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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31
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Nassal M, Rieger A. A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis. J Virol 1996; 70:2764-73. [PMID: 8627750 PMCID: PMC190133 DOI: 10.1128/jvi.70.5.2764-2773.1996] [Citation(s) in RCA: 124] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] 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. Encapsidation of this RNA pregenome is mediated by the interaction of the viral replication enzyme P with the structured 5'-proximal RNA element epsilon; replication was thought to start in the 3'-proximal direct repeat DR1*. However, recent data obtained with the duck hepatitis B virus indicated a novel, discontinuous mechanism of negative-strand DNA synthesis. Here we demonstrate, using DNA transfection of complete HBV genomes, that the 3'-half of a 6-nucleotide bulge in HBV epsilon whose primary sequence is not important for encapsidation serves as template for a short DNA primer that is subsequently transferred to DR1*. Apparently, P protein copies any template sequence that does not interfere with epsilon structure; however, altered primary sequences can induce polymerase stuttering, resulting in extended primers containing more than one equivalent of the template sequence. The importance of the bulged structure is emphasized by the dependence of primer length on bulge size. Transfer specificity is in part controlled by sequence complementarity. The strategy of using the 5' encapsidation signal as the origin of replication for discontinuous negative-strand DNA synthesis, common to mammalian and avian hepadnaviruses, suggests the evolutionary origin of hepatitis B viruses to lie between that of modern retroviruses and primitive retroelements like the Mauriceville retroplasmid.
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Affiliation(s)
- M Nassal
- Zentrum für Molekulare Biologie, Universität Heidelberg, Germany
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32
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Asahina Y, Enomoto N, Ogura Y, Sakuma I, Kurosaki M, Izumi N, Marumo F, Sato C. Complete nucleotide sequences of hepatitis B virus genomes associated with epidemic fulminant hepatitis. J Med Virol 1996; 48:171-8. [PMID: 8835351 DOI: 10.1002/(sici)1096-9071(199602)48:2<171::aid-jmv9>3.0.co;2-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Pre-core/core mutants are frequently observed in patients with fulminant hepatitis. To investigate the extent of molecular characteristics of hepatitis B virus (HBV) genomes implicated in the development of fulminant hepatitis, full-length HBV genomes were sequenced directly from sera of two patients with epidemic fatal fulminant hepatitis, after amplification by the polymerase chain reaction. These two genomes, of 3215 nucleotides, were 99.6% identical, indicating that a common source of HBV potentially caused fulminant hepatitis. Thirty unique nucleotide mutations were commonly found in the two entire HBV genomes. Three were located in the stem-loop structure, changing this element to a more stable structure. Twenty-five unique amino acid substitutions were found in each open reading frame, except for the X and pre-surface 2 genes. One was located in the pre-surface 1 gene; two were in the surface gene; three were in the pre-core gene, including codons 28 (tryptophan to stop codon) and 29 (glycine to aspartic acid); eight were in the core gene; and 11 were in the polymerase gene. The pre-core mutations at codons 28 and 29 were common to the two HBV strains reported previously in patients with epidemic fulminant hepatitis. Thus, HBV genomes associated with epidemic fatal fulminant hepatitis have numerous unique mutations, located mainly in the polymerase gene, as well as the pre-core/core gene, including mutations in the stem-loop structure of the pregenome encapsidation signal sequence. These mutations may be associated with the development of fulminant hepatitis.
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Affiliation(s)
- Y Asahina
- Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan
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33
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Affiliation(s)
- M Nassal
- Center for Molecular Biology, University of Heidelberg, Germany
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34
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Ton SH, Iskandar K, Noriah R, Thanaletchimy N. Nucleotide sequence of precore region of hepatitis B virus DNA in HBsAg-positive carriers in Malaysia. SCANDINAVIAN JOURNAL OF INFECTIOUS DISEASES 1996; 28:543-8. [PMID: 9060053 DOI: 10.3109/00365549609037957] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
As most published studies on precore mutants have been carried out on isolates from patients with liver diseases, and it is unclear whether HBsAg carriers with viraemia in the absence of HBeAg are also generally infected by such mutants, it was decided to sequence the precore region in some HBV-DNA isolated from HBsAg-positive carriers. Precore sequences of HBV-DNA from 43 HBsAg carriers in Malaysia were studied. Three HBV subtypes were identified according to the nucleotide sequence of the precore region. Most of the carriers were found to be infected by the subtype adr. Mutations were detected in the precore regions. The most common conserved mutation was a silent mutation involving conversion from T to C (CCT to CCC) at position 1858 at codon 15 (proline). It was found that 4/43 (9.3%) had a mutation at the penultimate codon where TGG was changed to TAG. All 4 isolates with the TAG mutation had nt T at position 1858. Of the 4 carriers who were infected by these mutant viruses, 2 were coinfected with the wild type, 1 was infected only by a variant with the mutation at position 1896, while another was infected by a variant with mutations at positions 1896 and 1899. Three of the 4 were anti-HBe positive while 1 was HBeAg positive. Alanine aminotransaminase activities in all 4 carriers were normal. This study therefore demonstrated that variants with stop codons at the penultimate codon could be found in asymptomatic carriers in Malaysia.
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Affiliation(s)
- S H Ton
- Department of Biochemistry, Medical Faculty, Universiti Kebangasaan Malaysia, Kuala Lumpur, Malaysia
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Zhang X, Zoulim F, Habersetzer F, Xiong S, Trépo C. Analysis of hepatitis B virus genotypes and pre-core region variability during interferon treatment of HBe antigen negative chronic hepatitis B. J Med Virol 1996; 48:8-16. [PMID: 8825704 DOI: 10.1002/(sici)1096-9071(199601)48:1<8::aid-jmv2>3.0.co;2-e] [Citation(s) in RCA: 97] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
The clinical importance of hepatitis B virus (HBV) genome variability has been reported recently. One example is the occurrence of hepatitis B virus pre-core mutants, which arise during spontaneous or interferon-induced seroconversion from HBeAg to anti-HBe and are thought to be selected by immune pressure. A survey of HBV pre-core mutants and viral genotypes in 35 HBeAg negative patients during interferon therapy was carried out to understand viral pathogenesis in this form of chronic hepatitis B. Seventeen patients responded to interferon therapy as assessed by the sustained normalization of serum ALT levels and the significant decrease of viremia levels. The response rate to interferon was independent of both initial serum viral DNA level and interferon doses. During interferon therapy, a significant decrease of M0 (wild-type pre-core sequence at pos. 1887-1908), M1 (TGG to TAG at pos. 1896) or M2 (TGG to TAG at pos. 1896, and GGC to GAC at pos. 1899) positive viral genomes was found in 48%, 42%, and 33% of patients, respectively. A higher response rate to interferon therapy was observed in patients infected with HBV genotype A (70%) or M0 positive strains (75%) as compared to patients infected with genotype D/E (40%) or M1/M2 positive strains (44%). The data support the hypothesis that pre-core defective HBV represent viral mutants with an increased capacity to resist exogenous alpha interferon. These findings emphasize that characterization of HBV genome variability prior to interferon therapy may help to predict antiviral response in HBeAg negative patients.
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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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Bahn A, Hilbert K, Martiné U, Westedt J, von Weizsäcker F, Wirth S. Selection of a precore mutant after vertical transmission of different hepatitis B virus variants is correlated with fulminant hepatitis in infants. J Med Virol 1995; 47:336-41. [PMID: 8636700 DOI: 10.1002/jmv.1890470408] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
The incidence of perinatal transmission of hepatitis B virus (HBV) depends on the HBeAg/anti-HBe status of the mother. While children of HBeAg-positive mothers have a 90% probability of acquiring a chronic hepatitis B virus carrier state, babies of anti-HBe-positive mothers are more likely to develop fulminant hepatitis within the first 3 to 4 months of life. There is evidence that precore (pre-C) mutations of the HBV can be associated with fulminant hepatitis. The pre-C region was therefore examined in sera from nine infants with fulminant hepatitis after vertical transmission, one HBeAg-positive and seven anti-HBe-positive mothers by polymerase chain reaction (PCR) and direct sequence analysis. In five mother/infant pairs the virus populations were characterized in addition by analysing clones of the amplified products. All mothers were infected with two or four variants of HBV with mutations at different positions of the preC genome including position 1896, which results in a stop codon. While the precore stop codon was detected in a portion of the virus populations of the HBeAg-positive and of four anti-HBe-positive mothers the dominating viral strain was represented by the wild type virus in three. In contrast, the virus populations of all babies showed the 1896 precore variant as the prevalent virus strain during the phase of active disease. In the surviving baby only wild type sequences were detected after recovery. Subtype ayw was found in all mothers and infants and adw2 was present in three mothers and in the surviving child. The findings suggest that all mothers carried a wild type HBV population with a certain number of different HBV variants. After transmission of the mixed virus population a selection process was started in the baby. The association of subtype ayw with the precore mutations and with the fatal outcome of the hepatitis B might be the result of a directed selection of this variant with a particular advantage in the viral life cycle.
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Affiliation(s)
- A Bahn
- Children's Hospital of the Johannes Gutenberg University, Mainz, Germany
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Rodriguez-Frias F, Buti M, Jardi R, Cotrina M, Viladomiu L, Esteban R, Guardia J. Hepatitis B virus infection: precore mutants and its relation to viral genotypes and core mutations. Hepatology 1995; 22:1641-7. [PMID: 7489968 DOI: 10.1002/hep.1840220605] [Citation(s) in RCA: 123] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The precore-core gene of hepatitis B virus (HBV) was directly sequenced from serum samples of 42 patients with chronic B hepatitis (19 hepatitis B e antigen [HBeAg]+ and 23 anti-HBE+). Viral genotypes were determined by comparison with 11 reference sequences and by restriction analysis. Genotype A was identified in 16 cases, genotype D in 24 cases, and other genotypes in 2 cases. Precore mutations, mainly M1 (stop at codon 28), were differently distributed among the viral genotypes: 3 cases (18.8%) with genotype A and 18 cases (75%) with genotype D. In sequences with precore mutants, the encapsidation signal was more stable (negative stabilization energy) than in sequences without precore mutants. In genotype A, the M1 mutation coexisted with a second mutation (C-->T at position 1858 in codon 15), and both mutations were paired in the secondary structure of the RNA encapsidation signal, which justified the rare presence of precore mutants in this genotype. The analysis showed different distribution of mutations depending on the viral genotype; patients with genotype D were more likely to have persistent HBV infection by selection of precore mutants. Multiple amino acid substitutions were detected in the core region, mainly in two subsequences that have been previously described as epitopes (flanked by codons 11 to 27 and 74 to 83); the presence of these mutations was significantly related to the presence of precore variants which abolished the expression of HBeAg.(ABSTRACT TRUNCATED AT 250 WORDS)
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Affiliation(s)
- F Rodriguez-Frias
- Liver Unit, Hospital General Universitario Valle Hebron, Barcelona, Spain
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Rieger A, Nassal M. Distinct requirements for primary sequence in the 5'- and 3'-part of a bulge in the hepatitis B virus RNA encapsidation signal revealed by a combined in vivo selection/in vitro amplification system. Nucleic Acids Res 1995; 23:3909-15. [PMID: 7479035 PMCID: PMC307309 DOI: 10.1093/nar/23.19.3909] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Hepatitis B virus (HBV) is a small DNA virus that replicates by reverse transcription of a terminally redundant RNA, the pregenome. Specific packaging of this transcript into viral capsids is mediated by interaction of the reverse transcriptase, P protein, with the 5'-proximal encapsidation signal epsilon, epsilon-function is correlated with the formation of a hairpin structure containing a bulge and a loop, each consisting of 6 nt. To analyse the importance of primary sequence in these regions, we have combined selection of encapsidation competent individuals from pools of randomized epsilon-sequences in transfected cells with in vitro amplification, thus bypassing the current experimental limitations of the HBV system. While no alterations of the authentic loop sequence were detectable, many different sequences were tolerated in the 3'-part of the bulge. However, at the two 5'-proximal bulge positions the wt sequence was strongly selected for, indicating that for RNA packaging close contacts between protein and the 5'- but not the 3'-part of the bulge are important. Such a bipartite organisation provides a structural basis for the recently demonstrated special role of the 3'-part of the bulge as template for the first nucleotides of (-)-strand DNA in HBV reverse transcription.
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Affiliation(s)
- A Rieger
- Zentrum für Molekulare Biologie, Universität Heidelberg, Germany
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