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Translational control mechanisms used by the human Hepatitis B virus : an upstream open reading frame modulates expression of the pregenomic RNAChen, Augustine, n/a January 2007 (has links)
The human hepatitis B virus (HBV) is a small hepatotropic virus, which affects approximately 350 million chronic sufferers worldwide. It has a compact 3.2 kbp dsDNA genome encoding four major overlapping genes namely core, polymerase, surface and X required for its replication. The virus synthesises a pregenomic RNA (pgRNA) which functions both as an RNA intermediate for reverse transcription into the DNA genome and as the mRNA for the translation of the core (C) and polymerase (P) proteins. The core overlaps the polymerase gene and is translated at a 10 to 1 ratio. The polymerase gene translated from the P AUG codon is preceded by at least 4 upstream AUG codons (uAUGs), namely C AUG, C1 AUG, J AUG and C2 AUG. Various mechanisms have been implicated in the synthesis of the polymerase protein. This led to the currently accepted model which involves leaky scanning and a reinitiation mechanism in polymerase synthesis.
However, multiple sequence alignment of the pgRNA revealed a short upstream open reading frame (uORF) highly conserved at the nucleotide level in all HBV subtypes and mammalian hepadnaviruses. This previously unreported uORF, designated as C0 ORF in this study is also conserved in its position and length. Past studies have either omitted this uORF in their test constructs or ignored its potential role. The C0 ORF has a conserved weak initiation context and is located within the epsilon structure within the 5� leader of the pgRNA, required for viral encapsidation. Importantly, the C0 ORF precedes and overlaps the core ORF, which may suggest an alternative model in which the core and polymerase may be translated and coordinately regulated.
Fusion of the C0 ORF to luciferase showed for the first time that this uORF is translated through the detection of reporter activity (~20% of C) and also visualisation of the fusion protein via western analysis using anti-C0 and anti-luciferase antibodies. Subsequent removal of the C0 ORF implicated a role in repressing downstream core fusion protein synthesis in HepG2 cells. A similar repression was observed on J expression.
To study the effect of C0 on downstream polymerase translation, a pgRNA-like DNA construct was made and subsequent mutations introduced. Mutation of the C0 AUG led to an increase in initiation at the downstream P AUG. Alteration of the existing weak initiation context to an optimal context which favours stronger initiation consistently showed a potential role for C0 ORF in facilitating reinitiation at certain downstream initiation codons including P AUG. Mutations of other uAUGs preceding the P AUG were also done to better understand their roles in regulating polymerase synthesis. The removal of the C AUG markedly increased expression from the P AUG. This study revealed other internal uAUGs in-frame to the C AUG, namely the C1 and C2 AUGs are also effectively translated, further reducing availability of translating ribosomes to downstream P AUG. Indeed the removal of the C1 and C2 AUGs led to a corresponding increase in initiation from the P AUG. Initiation at the internal J AUG was also reported and its removal showed a significant decrease in expression from the P AUG, consistent with the previous model implicating reinitiation at the P initiation site after translation of the short J ORF. The inhibitory role of the 5 uAUGs prior to the P AUG were confirmed when all were removed, giving rise to translation almost equal to that at C AUG.
Taken together, these results suggest a new model in which the HBV C0 ORF plays a key role in controlling core and polymerase synthesis by repressing core translation and making available more ribosomes to downstream AUGs possibly facilitating translation reinitiation. In addition, the translation of the C0 ORF across the [epsilon] region may also preclude encapsidation, potentially acting as a switch discriminating the pgRNA template between encapsidation and translation. Therefore, the highly conserved [epsilon] region and C0 ORF present an excellent target for molecular based antiviral drugs (antisense oligonucleotides, aptamers, ribozymes) potentially providing new anti HBV drugs.
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Hepatitis B virus in silvery gibbons (Hylobates moloch) /Payne, Karen Louise. January 2004 (has links)
Thesis (M.Phil.)--Murdoch University, 2004. / Thesis submitted to the Division of Veterinary and Biomedical Sciences. "A dissertation submitted to Murdoch University in fulfilment of the requirements for the degree of Masters in Philosophy in Zoo and Wildlife Medicine" Bibliography: leaves 134-143.
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Chronic hepatitis B-related liver diseases in the Chinese /Lai, Ching-lung. January 1993 (has links)
Thesis (M.D.)--University of Hong Kong, 1994. / Includes bibliographical references (leaves 239-283).
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Cell permeable nucleocapsids as a novel tool for efficient gene tranBrandenburg, Boerries. January 2005 (has links)
Berlin, Freie Univ., Diss., 2005. / Dateiforamt: zip, Dateien im PDF-Format. Computerdatei im Fernzugriff.
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Cell permeable nucleocapsids as a novel tool for efficient gene transfer & HBV biologyBrandenburg, Boerries. January 2005 (has links)
Berlin, Freie University, Diss., 2005. / Dateiforamt: zip, Dateien im PDF-Format.
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Chronic hepatitis B-related liver diseases in the ChineseLai, Ching-lung. January 1993 (has links)
Thesis (M.D.)--University of Hong Kong, 1994. / Includes bibliographical references (leaves 239-283) Also available in print.
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Untersuchungen zur posttranslationalen präS-Translokation des grossen Hüllproteins des Hepatitis-B-VirusLambert, Carsten. January 2001 (has links) (PDF)
Mainz, Univ., Diss., 2001.
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Nucleic acid sequence analysis of the distal X gene region containing the basic core promoter of the hepatitis B virus in southern African asymptomatic carriers of the virus and hepatocellular carcinoma patientsBaptista, Marina Da Conceicao Pinto Azevedo 07 March 2014 (has links)
The purpose of this study was to identify mutations in the basic core promoter and
enhancer II region a* the hepatitis B virus (HBV) that might result in the hepatitis B virus
e antigen (HBeAg)-negative phenotype and contribute to hepatocarcinogenesis in black
African carriers of the virus. The basic core promoter/enhancer II overlaps the X gene.
HBV DNAfrom serum of 47 asymptomatic carriers and 50 patients with hepatocellular
carcinoma and from 29 tumorous and 10 nontumorous liver tissues was amplified and
sequenced directly. That part of the enhancer II region not overlapping the basic core promoter was reasonably well conserved in all samples. Missense mutations at
positions 1809 and 1812 were found in 80% of all sequences and may represent
wiidtype sequence in southern African isolates. Nucleotide and amino acid divergences
were higher in the basic core promoter of hepatocellular carcinoma patients than of
asymptomatic carriers (p<0.0001). This applied particularly to the paired 1762 adenine
to thymine (1762T) and 1764 guanine to adenine (1764*) missense mutations, the prevalence of which was 66% in patients with hepatocellular carcinoma compared with
11% in asymptomatic carriers (p<0.0001). There was no association between the presence of 1762T1764A and the rate of HBeAg negativity, although these mutations
suppressed HBeAg titres in HBeAg-positive patients. Suppression of HBeAg expression
as well as alteration of amino acid sequence of the X protein may be contributing factors
in the development of hepatocarcinogenesis.
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Helper-dependent adenoviral vectors expressing anti-HBV pri-miR sequences from the liver-specific PEPCK promoterSmit, Duodane January 2017 (has links)
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree
of
Master of Science in Medicine
Johannesburg, 2017 / Hepatitis B is a global health problem that kills about 600 000 people annually. It is an infectious disease caused by the Hepatitis B Virus (HBV), which infects the liver and leads to liver inflammation and secondary complications such as cirrhosis and Hepatocellular Carcinoma (HCC). The available therapies are only partially effective and are associated with adverse side effects and viral drug resistance. RNA interference (RNAi) pathway is a gene silencing pathway found in diverse living systems including mammals. Harnessing of this pathway to inhibit HBV replication has shown a lot of promise, with highly effective anti-HBV RNAi activator sequences designed. However, the lack of safe and efficient delivery and expression systems for these sequences is one of the obstacles that need to be overcome before RNAi effectors can reach clinical application. For easy assessment of transduction efficiency, Helper Dependent Adenoviral vectors (HDAd) expressing β-galactosidase (encoded by lac Z gene) are commonly used to deliver anti-viral RNAi activators. However, this reporter protein has been blamed for induction of innate immune response and concomitant clearance of the HDAds by the host. For the first time, this study explored the use of lac Z-deficient HDAds to deliver anti-HBV RNAi activators expressed under the control of a liver-specific phosphoenolpyruvate carboxykinase (PEPCK) promoter. HDAd expressing Firefly luciferase resulted in a significant luminescence detected in cell culture lysates and a sustained bioluminescence in mice. HDAds expressing anti-HBV sequences transduced the liver efficiently and did not induce a pronounced inflammatory response or liver toxicity in mice. However, this did not translate into maximal anti-HBV sequence expression and HBV replication inhibition in vitro and in vivo. This suggests that the PEPCK promoter is inadequate for RNAi activator expression. This study highlights the importance of careful RNAi activator regulatory elements selection and presents the therapeutic potential and utility of HDAd vectors for hepatotropic delivery of antiviral sequences with markedly attenuated immune response stimulation and toxicity. For improved anti-HBV RNAi activator expression and HBV knockdown, a different liver specific promoter mouse transthyretin receptor (MTTR) promoter is currently being investigated in our lab. / MT2017
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INHIBITING HEPATITIS B VIRUS GENE EXPRESSION WITH HAMMERHEAD RIBOZYMES THAT TARGET THE HBx OPEN READING FRAMEWeinberg, Marc Saul 28 October 2002 (has links)
A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the
degree of
Doctor of Philosophy
Johannesburg, 2002 / Hepatitis B virus (HBV) infection is endemic to several populous regions and is often complicated by cirrhosis and hepatocellular carcinoma (HCC). Present treatment of chronic HBV infection is usually ineffective and novel therapeutic approaches are an important medical objective. The X open reading frame (ORF) of HBV, HBx, is a conserved sequence that overlaps with the polymerase ORF and viral c/'s-elements, and is present within all viral transcripts. In addition, the HBx ORF encodes a 17 kDa transactivator protein, HBx, which is required for the establishment of viral infection and has been implicated in HBV-associated hepatocarcinogenesis. The HBx sequence thus represents a compelling target for applying nucleic acid hybridisation-based therapeutic agents for the inhibition of HBV gene expression and replication. / IT2018
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