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van den Berg F, Limani SW, Mnyandu N, Maepa MB, Ely A, Arbuthnot P. Advances with RNAi-Based Therapy for Hepatitis B Virus Infection. Viruses 2020; 12:E851. [PMID: 32759756 PMCID: PMC7472220 DOI: 10.3390/v12080851] [Citation(s) in RCA: 44] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 07/22/2020] [Accepted: 07/29/2020] [Indexed: 02/06/2023] Open
Abstract
Infection with hepatitis B virus (HBV) remains a global health challenge. Approximately 292 million people worldwide are chronically infected with HBV and the annual mortality from the infection is approaching 900,000. Despite the availability of an effective prophylactic vaccine, millions of individuals are at risk of potentially fatal complicating cirrhosis and hepatocellular carcinoma. Current drug treatments can suppress viral replication, slow the progression of liver fibrosis, and reduce infectivity, but can rarely clear the viral covalently closed circular DNA (cccDNA) that is responsible for HBV persistence. Alternative therapeutic strategies, including those based on viral gene silencing by harnessing the RNA interference (RNAi) pathway, effectively suppress HBV replication and thus hold promise. RNAi-based silencing of certain viral genes may even lead to disabling of cccDNA during chronic infection. This review summarizes different RNAi activators that have been tested against HBV, the advances with vectors used to deliver artificial potentially therapeutic RNAi sequences to the liver, and the current status of preclinical and clinical investigation.
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Affiliation(s)
| | | | | | | | | | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2050, South Africa; (F.v.d.B.); (S.W.L.); (N.M.); (M.B.M.); (A.E.)
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Wettengel JM, Burwitz BJ. Innovative HBV Animal Models Based on the Entry Receptor NTCP. Viruses 2020; 12:E828. [PMID: 32751581 PMCID: PMC7472226 DOI: 10.3390/v12080828] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2020] [Revised: 07/24/2020] [Accepted: 07/27/2020] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B is a major global health problem, with an estimated 257 million chronically infected patients and almost 1 million deaths per year. The causative agent is hepatitis B virus (HBV), a small, enveloped, partially double-stranded DNA virus. HBV has a strict species specificity, naturally infecting only humans and chimpanzees. Sodium taurocholate co-transporting polypeptide (NTCP), a bile acid transporter expressed on hepatocytes, has been shown to be one of the key factors in HBV infection, playing a crucial role in the HBV entry process in vitro and in vivo. Variations in the amino acid sequence of NTCP can inhibit HBV infection and, therefore, contributes, in part, to the species barrier. This discovery has revolutionized the search for novel animal models of HBV. Indeed, it was recently shown that variations in the amino acid sequence of NTCP represent the sole species barrier for HBV infection in macaques. Here, we review what is known about HBV entry through the NTCP receptor and highlight how this knowledge has been harnessed to build new animal models for the study of HBV pathogenesis and curative therapies.
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Affiliation(s)
- Jochen M. Wettengel
- Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Trogerstr. 30, 81675 Munich, Germany;
- Vaccine & Gene Therapy Institute, Oregon Health & Science University, 505 N.W. 185th Avenue Beaverton, Tanasbourne, OR 97006, USA
| | - Benjamin J. Burwitz
- Vaccine & Gene Therapy Institute, Oregon Health & Science University, 505 N.W. 185th Avenue Beaverton, Tanasbourne, OR 97006, USA
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In Vitro Systems for Studying Different Genotypes/Sub-Genotypes of Hepatitis B Virus: Strengths and Limitations. Viruses 2020; 12:v12030353. [PMID: 32210021 PMCID: PMC7150782 DOI: 10.3390/v12030353] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2020] [Revised: 03/04/2020] [Accepted: 03/06/2020] [Indexed: 12/11/2022] Open
Abstract
Hepatitis B virus (HBV) infects the liver resulting in end stage liver disease, cirrhosis, and hepatocellular carcinoma. Despite an effective vaccine, HBV poses a serious health problem globally, accounting for 257 million chronic carriers. Unique features of HBV, including its narrow virus-host range and its hepatocyte tropism, have led to major challenges in the development of suitable in vivo and in vitro model systems to recapitulate the HBV replication cycle and to test various antiviral strategies. Moreover, HBV is classified into at least nine genotypes and 35 sub-genotypes with distinct geographical distributions and prevalence, which have different natural histories of infection, clinical manifestation, and response to current antiviral agents. Here, we review various in vitro systems used to study the molecular biology of the different (sub)genotypes of HBV and their response to antiviral agents, and we discuss their strengths and limitations. Despite the advances made, no system is ideal for pan-genotypic HBV research or drug development and therefore further improvement is required. It is necessary to establish a centralized repository of HBV-related generated materials, which are readily accessible to HBV researchers, with international collaboration toward advancement and development of in vitro model systems for testing new HBV antivirals to ensure their pan-genotypic and/or customized activity.
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Ruan J, Ping CY, Sun S, Cheng X, Han PY, Zhang YG, Sun DX. Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines. World J Gastroenterol 2019; 25:5961-5972. [PMID: 31660033 PMCID: PMC6815792 DOI: 10.3748/wjg.v25.i39.5961] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/19/2019] [Revised: 08/08/2019] [Accepted: 09/13/2019] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Previously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research.
AIM To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.
METHODS We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.
RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity.
CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.
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Affiliation(s)
- Jie Ruan
- The Liver Disease Center of Chinese People’s Liberation Army, the 980th Hospital of Chinese People’s Liberation Army Joint Logistics Support Force, Shijiazhuang 050082, Hebei Province, China
- Department of Infection and Liver Disease, Shannxi University of Chinese Medicine Affiliated Hospital, Xianyang 712000, Shannxi Province, China
| | - Cai-Yan Ping
- The Liver Disease Center of Chinese People’s Liberation Army, the 980th Hospital of Chinese People’s Liberation Army Joint Logistics Support Force, Shijiazhuang 050082, Hebei Province, China
| | - Shuo Sun
- The Liver Disease Center of Chinese People’s Liberation Army, the 980th Hospital of Chinese People’s Liberation Army Joint Logistics Support Force, Shijiazhuang 050082, Hebei Province, China
| | - Xin Cheng
- The Liver Disease Center of Chinese People’s Liberation Army, the 980th Hospital of Chinese People’s Liberation Army Joint Logistics Support Force, Shijiazhuang 050082, Hebei Province, China
| | - Peng-Yu Han
- The Liver Disease Center of Chinese People’s Liberation Army, the 980th Hospital of Chinese People’s Liberation Army Joint Logistics Support Force, Shijiazhuang 050082, Hebei Province, China
| | - Yin-Ge Zhang
- The Liver Disease Center of Chinese People’s Liberation Army, the 980th Hospital of Chinese People’s Liberation Army Joint Logistics Support Force, Shijiazhuang 050082, Hebei Province, China
| | - Dian-Xing Sun
- The Liver Disease Center of Chinese People’s Liberation Army, the 980th Hospital of Chinese People’s Liberation Army Joint Logistics Support Force, Shijiazhuang 050082, Hebei Province, China
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Schreiner S, Nassal M. A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation-and Beyond? Viruses 2017; 9:v9050125. [PMID: 28531167 PMCID: PMC5454437 DOI: 10.3390/v9050125] [Citation(s) in RCA: 69] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2017] [Revised: 05/16/2017] [Accepted: 05/18/2017] [Indexed: 12/12/2022] Open
Abstract
Chronic hepatitis B virus (HBV) infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg) RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC) DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc) DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR), a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system.
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Affiliation(s)
- Sabrina Schreiner
- Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Ingolstädter Landstr. 1, Neuherberg, D-85764 Munich, Germany.
| | - Michael Nassal
- Dept. of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany.
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Liu X, Yuan L, Yuan Q, Zhang Y, Wu K, Zhang T, Wu Y, Hou W, Wang T, Liu P, Shih JWK, Cheng T, Xia N. Detection and analysis of tupaia hepatocytes via mAbs against tupaia serum albumin. Exp Anim 2015; 65:117-23. [PMID: 26597317 PMCID: PMC4873480 DOI: 10.1538/expanim.15-0086] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
On the basis of its close phylogenetic relationship with primates, the development of
Tupaia belangeri as an infection animal model and drug metabolism model
could provide a new option for preclinical studies, especially in hepatitis virus
research. As a replacement for primary human hepatocytes (PHHs), primary tupaia
hepatocytes (PTHs) have been widely used. Similar to human serum albumin, tupaia serum
albumin (TSA) is the most common liver synthesis protein and is an important biomarker for
PTHs and liver function. However, no detection or quantitative method for TSA has been
reported. In this study, mouse monoclonal antibodies (mAbs) 4G5 and 9H3 against TSA were
developed to recognize PTHs, and they did not show cross-reactivity with serum albumin
from common experimental animals, such as the mouse, rat, cow, rabbit, goat, monkey, and
chicken. The two mAbs also exhibited good performance in fluorescence activated cell
sorting (FACS) analysis and immunofluorescence (IF) detection of PTHs. A chemiluminescent
enzyme immune assay method using the two mAbs, with a linear range from 96.89 pg/ml to
49,609.38 pg/ml, was developed for the quantitative detection of TSA. The mAbs and the
CLEIA method provide useful tools for research on TSA and PTHs.
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Affiliation(s)
- Xuan Liu
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, 361102, P.R. China
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Ramanan V, Scull MA, Sheahan TP, Rice CM, Bhatia SN. New Methods in Tissue Engineering: Improved Models for Viral Infection. Annu Rev Virol 2014; 1:475-499. [PMID: 25893203 PMCID: PMC4398347 DOI: 10.1146/annurev-virology-031413-085437] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
New insights in the study of virus and host biology in the context of viral infection are made possible by the development of model systems that faithfully recapitulate the in vivo viral life cycle. Standard tissue culture models lack critical emergent properties driven by cellular organization and in vivo-like function, whereas animal models suffer from limited susceptibility to relevant human viruses and make it difficult to perform detailed molecular manipulation and analysis. Tissue engineering techniques may enable virologists to create infection models that combine the facile manipulation and readouts of tissue culture with the virus-relevant complexity of animal models. Here, we review the state of the art in tissue engineering and describe how tissue engineering techniques may alleviate some common shortcomings of existing models of viral infection, with a particular emphasis on hepatotropic viruses. We then discuss possible future applications of tissue engineering to virology, including current challenges and potential solutions.
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Affiliation(s)
- Vyas Ramanan
- Harvard-MIT Division of Health Sciences and Technology, Institute for Medical Engineering and Science, Cambridge, Massachusetts 02139
| | - Margaret A Scull
- Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065
| | - Timothy P Sheahan
- Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065
| | - Charles M Rice
- Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065
| | - Sangeeta N Bhatia
- Harvard-MIT Division of Health Sciences and Technology, Institute for Medical Engineering and Science, Cambridge, Massachusetts 02139
- Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
- Division of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
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Wang Z, Wu L, Cheng X, Liu S, Li B, Li H, Kang F, Wang J, Xia H, Ping C, Nassal M, Sun D. Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation. PLoS One 2013; 8:e60306. [PMID: 23589756 PMCID: PMC3615001 DOI: 10.1371/journal.pone.0060306] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2012] [Accepted: 02/25/2013] [Indexed: 12/13/2022] Open
Abstract
Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better understand, and combat, this important pathogen.
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Affiliation(s)
- Zihua Wang
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
- The Third Military Medical University, Chongqing, PR China
| | - Li Wu
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
| | - Xin Cheng
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
| | - Shizhu Liu
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
| | - Baosheng Li
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
| | - Haijun Li
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
| | - Fubiao Kang
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
| | - Junping Wang
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
| | - Huan Xia
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
| | - Caiyan Ping
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
| | - Michael Nassal
- University Hospital Freiburg, Internal Medicine II/Molecular Biology, Freiburg, Germany
- * E-mail: (DS); (MN)
| | - Dianxing Sun
- The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, PR China
- * E-mail: (DS); (MN)
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Dandri M, Lütgehetmann M, Petersen J. Experimental models and therapeutic approaches for HBV. Semin Immunopathol 2012; 35:7-21. [PMID: 22898798 DOI: 10.1007/s00281-012-0335-7] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2012] [Accepted: 07/31/2012] [Indexed: 12/12/2022]
Abstract
Liver disease associated to persistent infection with the hepatitis B virus (HBV) continues to be a major health problem of global impact. In spite of the existence of an effective vaccine, approximately 360 million people are chronically infected worldwide, who are at high risk of developing liver cirrhosis and hepatocellular carcinoma. Although current therapeutic regimens can efficiently suppress viral replication, the unique replication strategies employed by HBV permit the virus to persist within the infected hepatocytes. As a consequence, relapse of viral activity is commonly observed after cessation of treatment with polymerase inhibitors. The narrow host range of HBV has hindered progresses in understanding specific steps of HBV replication and the development of more effective therapeutic strategies aiming at achieving sustained viral control and, eventually, virus eradication. This review will focus on summarizing recent advances obtained with well-established and more innovative experimental models, giving emphasis on the strength of the different systems as tools for elucidating distinct aspects of HBV persistence and for the development of new therapeutic approaches.
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Affiliation(s)
- Maura Dandri
- Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
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HBV life cycle: entry and morphogenesis. Viruses 2009; 1:185-209. [PMID: 21994545 PMCID: PMC3185491 DOI: 10.3390/v1020185] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2009] [Revised: 07/31/2009] [Accepted: 08/13/2009] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B virus (HBV) is a major cause of liver disease. HBV primarily infects hepatocytes by a still poorly understood mechanism. After an endocytotic process, the nucleocapsids are released into the cytoplasm and the relaxed circular rcDNA genome is transported towards the nucleus where it is converted into covalently closed circular cccDNA. Replication of the viral genome occurs via an RNA pregenome (pgRNA) that binds to HBV polymerase (P). P initiates pgRNA encapsidation and reverse transcription inside the capsid. Matured, rcDNA containing nucleocapsids can re-deliver the RC-DNA to the nucleus, or be secreted via interaction with the envelope proteins as progeny virions.
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Roberts TE, Sargis EJ, Olson LE. Networks, trees, and treeshrews: assessing support and identifying conflict with multiple loci and a problematic root. Syst Biol 2009; 58:257-70. [PMID: 20525582 PMCID: PMC2715937 DOI: 10.1093/sysbio/syp025] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2008] [Revised: 12/09/2008] [Accepted: 02/18/2009] [Indexed: 11/13/2022] Open
Abstract
Multiple unlinked genetic loci often provide a more comprehensive picture of evolutionary history than any single gene can, but analyzing multigene data presents particular challenges. Differing rates and patterns of nucleotide substitution, combined with the limited information available in any data set, can make it difficult to specify a model of evolution. In addition, conflict among loci can be the result of real differences in evolutionary process or of stochastic variance and errors in reconstruction. We used 6 presumably unlinked nuclear loci to investigate relationships within the mammalian family Tupaiidae (Scandentia), containing all but one of the extant tupaiid genera. We used a phylogenetic mixture model to analyze the concatenated data and compared this with results using partitioned models. We found that more complex models were not necessarily preferred under tests using Bayes factors and that model complexity affected both tree length and parameter variance. We also compared the results of single-gene and multigene analyses and used splits networks to analyze the source and degree of conflict among genes. Networks can show specific relationships that are inconsistent with each other; these conflicting and minority relationships, which are implicitly ignored or collapsed by traditional consensus methods, can be useful in identifying the underlying causes of topological uncertainty. In our data, conflict is concentrated around particular relationships, not widespread throughout the tree. This pattern is further clarified by considering conflict surrounding the root separately from conflict within the ingroup. Uncertainty in rooting may be because of the apparent evolutionary distance separating these genera and our outgroup, the tupaiid genus Dendrogale. Unlike a previous mitochondrial study, these nuclear data strongly suggest that the genus Tupaia is not monophyletic with respect to the monotypic Urogale, even when uncertainty about rooting is taken into account. These data concur with mitochondrial DNA on other relationships, including the close affinity of Tupaia tana with the enigmatic Tupaia splendidula and of Tupaia belangeri with Tupaia glis. We also discuss the taxonomic and biogeographic implications of these results.
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Affiliation(s)
- Trina E Roberts
- University of Alaska Museum and Institute of Arctic Biology, University of Alaska-Fairbanks, Fairbanks, AK 99775, USA.
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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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