Ai)ë' AND HEPATITIS DELTA VIRUS. Tom Bernard MACNAUGHTON B. Sc (Hons) Division of Medical Virology lnstitute of Medical and Veterinary Science ADEI.AIDE. South Australia A thesis submitted to the University of Adelaide in fulfilment of the requirements for the degree of Doctor of Philosophy December 1990. CONTENTS. SU M MARY DECLARATION ACKNOWLEDG MENTS PUBLICATIONS RESULTING AB BREVIATIONS CHAPTER f : INTRODUCTION PREFACE. 1.1 oVERVIEW. A Hepatitis B Virus: Historical Perspectives. B Pathogenesis of HBV lnfection. C Animal Models of HBV. D ln Vitro Models of Hepadnavirus lnfection. 1.2 PARTICLES ASSOCIATED WITH HBV INFECTION. A Filaments and Spheres. B Hepatitis B Virion. 1.3 HEPADNAVIRUS GENOME ORGANISATION. A Genome Structure B Open Reading Frames and Their Protein Products. C Control Elements and RNA Transcription. 1.4 REPLICATION STRATEGY OF HEPADNAVIRUSES. A Adsorption. B Uncoating to Synthesis of cccDNA. C RNA Synthesis. D DNA Synthesis. E Envelopment and Secretion. F lntracellular Amplification of cccDNA. 1.5 HEPATITIS DELTA VIRUS. A Overview. B HDV RNA. C Hepatitis Delta Antigen. D HDV RNA Replication. 1.6 AIMS OF THESIS. CHAPTER 2: MATERIALS AND METHODS. 2.1 EUKARYOTIC CELL CULTURES. A Media. B Culture Methods. C Nucleotide lncorporation Assays. 2.2 PROKARYOTIC CELL CULTURE. A Media and Cells. B Preparation of Competent Cells. ¡i 2.3 CLONING. A Vec'tors and Plasmids. B Phenol Extraction and Ethanol Precipitation of Nucleic acids. C DNA Modifying Enzymes. D Agarose Gel Electrophoresis. E Transformation and Plasmid Amplification. 2.4 TRANSFECTION. A Adherent Cell Technique. B Suspension Cell Technique. C RNA Transfection. D Subcloning. E G418 Titration. 2.5 NUCLEIC ACID HYBRIDISATION. A Sample Preparation. B Transfer of Nucleic Acids to Nitrocelluose. C Dot Blots. D Nucleic Acid Probes. E Hybridisation. 2.6 ANTIGEN DETECTION. A lmmunofluorescence. B ELISA. C RIA. D lmmunoblotting. 2.7 INFECTION AND SECRETION EXPERIMENTS. A Screening of lnocula. B Purification of HB Virions. C lnfection of Cultured Cells. D HBsAg Secretion Assays. E Duck lnoculation. 2.8 RNA TRANSCRIPTION INHIBITION ASSAYS. A Actinomycin D. B s-Amanitin. C Transcription from Exogenously-Added RNA Templates. 2.9 ANALYSIS OF RECOMBINANT HDAg. A Purification of Recombinant HDAg. B HPLC Fractionation. C Ultracentrifugation. CHAPTER 3: tÂl VTTRO CULTURE OF HBV. 3.1 INTRODUCTION. 3.2 EXPERIMENTAL DESIGN. A Cell Lines. 3.3 RESULTS. A Selection of HBV DNA Positive Sera as lnocula. B lnfection Experiments. 3.4 DrscussroN. A. Block in Events Leading to HBV lnfection ln Vitro. B Factors that lnfluence Adsorption of HBV to Cultured Cells. C HBV RNA Transcription. CHAPTER 4: HBV ANTIGEN EXPRESSION IN HEPATOMA.DERIVED AND TRANSFECTED CELL LINES, 4.1 INTRODUCTION. 4.2 EXPERIMENTAL METHODS AND DESIGN. A Cell Lines. B Detection of HBV-Specific Markers. 4.3 RESULTS. A Methylation Patterns of lntegrated HBV DNA in TK4 Clones A1 and 84. B HBsAg Secretion. (a) Hormones. (b) DMEM vs LHC-4 media. (c) Etfect of calcium on HBsAg secretion. C Effect of Demethylation. (a) TK4 Clone 41. (b) TK4 Clone 84. (c) Other cell lines. D The Effect of Calcium on HBV Gene Expression. (a) H BsAg-specific protei ns. (b) HBcAg. (c) HBV production. E HBV lnfection. 4.4 DrscussloN. A Regulatory Elements of the HBsAg Gene. B Cytosine Methylation. C Etfect of Calcium. D The Effect of Glucagon. E Conclusion. CHAPTER 5: HEPADNAVIRUS TRANSFECTION. 5.1 INTRODUCTION AND EXPERIMENTAL DESIGN. 5.2 RESULTS. A PlasmidConstruction. B Production of Stable Cell Lines. (a) DHBV antigen expression. (b) DHBV antigen stain¡ng in G2 DHBV-3 cells. (c) Replicative intermediates in G2 DHBV-3 cells. (d) Recloning of G2 DHBV-3 cells. C ln Vítro Synthesis of lnfectious DHBV. s.3 DrscussloN. CHAPTER 6: EXPRESSION, PROPERTIES AND APPLICATIONS OF RECOMBINANT HDAg. 6.1 INTRODUCTION. 6.2 EXPERIMENTAL METHODS. A Source of HDV cDNA for Cloning. B Expression Studies. tv 6.3 RESULTS. A Construction of Plasmids. B Detection of rHDAg in Transfected Cells. (a) lmmunofluorescence. (b) Radioimmunoassay. (c) lmmunoblotting. c Physical Properties of rHDAg. (a) HPLC analysís. (b) Density of rHDAg. (c) Sedimentation value of rHDAg. D RNA-Binding Properties of rHDAg. E Actinomycin D Studies. F Diagnostic Applications of rHDAg. 6.4 DrscussroN. CHAPTER 7: HEPATITIS DELTA VIRUS RNA REPLICATION. 7.1 INTRODUCTION. 7.2 AIMS AND EXPERIMENTAL METHODS. 7.3 RESULTS. A Construction of Plasmids. B Expression and Localisation of HDAg in Transfected HH1 Cells. C lntegration of HDV cDNA. D Localisation and Configuration of HDV RNA in H1ô8 and H1õ9 Cells. (a) In Situ cytohybridisation. (b) Northern blot hybridisation. (c) Polyadenylated species of HDV RNA. (d) Double stranded HDV RNA. E Mechanism of HDV RNA Synthesis. (a) Actinomycin D. (b) cr-Amanitin. F HDV RNA Synthesis: The Role of HDAg. G H1ô8 and H1õ9 Cells as a Model for Persistent HDV lnfection. (a) Effect of H1ô9 cell passage number on HDV RNA synthesis and expression. (b) Comparison of the HDAg polypeptide proflles in the 43, H1õ8 and H1õ9 cell lines. (c) HDAg polypeptide profiles in HDV-infected liver. H HBV Helper Function. (a) HDV replicat¡on. (b) HDV encapsidation and secretion. 7.4 DISCUSSION. A HDV RNA Replication in the H1õ8 and H1ô9 Cell Lines. B The ldentity of the HDV RNA Replicase. c HDV-Related Cytotoxicity. D Mechanism of HDAg-pz4 and -p27 Synthesis. E HBV Helper Function. V CHAPTER 8: CONCLUDING REMARKS. 8.1 TNTRODUCTION. 8.2 TRANSFECTION. 8.3 HEPADNAVTRUS CULTURE AND GENE EXPRESSION l[ vtTRo. A lnfection. B Future Prospects for HBV Replication in Continuous Cell Lines. 8.4 HEPATITIS DELTA V¡RUS. A Cell Specificity. B HBV Helper Function. C Properties of HDAg. D HBV Interference. E HDV RNA Replícation and Gene Expression. F Replication Strategy of HDV. 8.5 CONCLUSION. REFE R ENCES. APPEN DIX. PUBLICATIONS RESULTING FROM THIS WORK. vi SU MMARY. Hepadnavirus and HDV replication and gene expression were examined, with particular emphasis on the block(s) preventing HBV infection ¡IuiÍo, the extent of the helper function provided to HDV by HBV and the mechanism of HDV RNA replication. ATTEMPTS TO CULTURE HBV IN CONTINUOUS CELL LINËS. A total of 23 primary and continuous cell lines of human and animal origin, including 5 derived from human hepatomas, were evaluated for their ability to support HBV infection in vitro. lnfection protocols included the manipulation of classic variables, viz. virus concentration, and adsorption time and temperature, and examined the effecls of hormones and substances that promote gene deregulation or cellular differentiation. However, despite these attempts, in vitro HBV infection was not achieved. Consequently, factors which regulate HBV protein synthesis were then examined in the hope that this might in itself provide clues to HBV cultivation in vitro. These studies showed that steroid hormones stimulated HBsAg production, low calcium medium increased levels of secreted HBsAg and HBV, and S-azacytidine treatment induced HBcAg synthesis. However manipulation of these factors still did not lead to successful cultivation of HBV. SYNTHESIS OF INFECTIOUS DHBV IN A HUMAN HEPATOMA CELL LINE. To examine the possibility that the HepG2 cell line, known to support HBV replicat¡on after transfection of HBV DNA could also support replication of a related hepadnavirus, a dimeric construct of DHBV DNA contained in the vector pSV2Neo was transfected into this cell line. All five clones that were successfully expanded expressed DHBsAg, and one (G2 DHBV-3) was also positive for DHB pre-S and DHBcAg and synthesised infectious DHBV. Nevertheless, despite the G2 DHBV-S cell line displaying a homogeneous pattern of integrated DHBV DNA, only a low vil percentage of virus antigen-positive cells were detected at passage #5 or greater, and approximately one tenth of the mature virus produced by HepG2 cells after transfection with HBV DNA was synthesised. Thus, although the hepadnavirus species barrier can be breached, efficient transcription and/or translation of DHBV mRNA was likely to depend on both hepatocyte- and species-specific factors. EXPRESSION AND PROPERTIES OF RECOMBINANT HDAg. The gene encoding the hepatitis delta virus structural antigen was linked to a neomycin resistance gene in a retrovirus expression vector, and human HepG2 and HeLa cells lransfected with the recombinant plasmid. Several clones showed varying percentages of recombinant HDAg (rHDAg)-positive cells but most of these were lost in culture suggesting that HDAg may be cytotoxic. Thus only one cell line (43) derived from HepG2 cells that expressed rHDAg in the nuclei ol 100o/o of cells was expanded successfully. The HDAg staining pattern in the A3 cell line indicated a close relationship with cell nucleoli. Analysis of partially-purified rHDAg by HPLC showed a molecular weight in the range of 7x102 -2x10 3 kD that appeared to contain conformational-dependent epitopes, while the density of the antigen was 1.199/cm3 by equilibrium centrifugation in caesium chloride, and in rate zonal centrifugation the antigen sedimented with a value of 50S, close to that of particulate HBsAg. lmmunoblotting demonstrated a single polypeptide with a molecular weight ol 24kD that corresponded to the smaller of the two HDAg-specific polypeptides present in infected sera. The rHDAg polypeptide was shown to be a RNA-binding protein with specificity for both genomic- and antigenomic-species of hepatitis delta virus RNA. The A3 cell line was also used successfully in an indirect immunofluorescence assay to detect anti-HD, and rHDAg extracted from these cells permitted the substitution of human liver-derived HDAg in a competitive anti-HD radioimmu noassay. vii¡ HDV RNA REPLICATION AND GENE EXPRESSION. Multimeric constructs of HDV cDNA were assembled and introduced into the same retrovirus expression vector used in the experiments described above. A recombinant plasmid, designed to produce trimeric-length HDV RNA of antigenomic-sense, was transfected into a subclone of the HBsAg-positive PLC/PRF/S hepatoma cell line, and two stable cell lines (H1ô8 and H1ô9) were selected and clonally amplified.