HEPATITIS B VIRUS GENE MUTATIONS ASSOCIATED WITH HBEAG SEROCONVERSION CHENG YAN. (Ms. Medicine Beijing Medical University, China) A THESIS SUBMITTED

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1 HEPATITIS B VIRUS GENE MUTATIONS ASSOCIATED WITH HBEAG SEROCONVERSION CHENG YAN (Ms. Medicine Beijing Medical University, China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MEDICINE FACULTY OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2003 i

2 ACKNOWLEDGMENT I would like to express my gratitude to my supervisors, Associated Professor Lim Seng Gee, Dr. Frank Joseph Peter and Dr. Tan May Chin, Theresa for being very understanding and supportive, and for their invaluable supervision and guidance, throughout my study. I also would like to thank Dr. Shanthi Wasser and Dr. Emma Jane Croager for their support and advice during the project. I am indebted to Dr Brusic Vladimir, Dr. Edwin Chan Shih-Yen, Dr. Jeremiah Stanson Joseph and Mr. Vinsensius Berlian Vega S N for their valuable assistance and advice in phylogenetic analysis in this study. I am grateful to the members of the Hepatology laboratory, especially Seet Bee Leng, Ong Shair Ling Carmen and Tasneem Shabbir Kajiji for their technical support, discussion and invaluable friendship. I acknowledge the National University of Singapore for the award of the research scholarship. I also would like to thank my friends for being there to share my happiness and distress and for their love and care. Last but not least, I would like to record my sincere thanks to my family for their endless support, encouragement, patience and love. This work was supported by research grants NMRC/C to A/Prof. Lim seng Gee from the National Research Council, Singapore and BMRC/R to Dr. Frank Joseph Peter from Biomedical Research Council, Singapore. ii

3 CONTENTS Acknowledgements Contents Summary Publications and conference abstract Abbreviations ii iii xiv xvi xvii CHAPTER 1 INTRODUCTION History The hepatitis B virus The life cycle of HBV Attachment and entry into the host cells HBV genome repair and transcription HBV gene translation Viral reverse transcription Virion formation Covalently closed circular DNA pool Hepatitis B virus particle The HBV genome HBV promoters and signal regions HBV Gene products Hepatitis B surface proteins Hepatitis B core and e proteins 23 iii

4 Hepatitis B polymerase protein Hepatitis B x protein HBV genotype and HBsAg subtype HBV: the disease Prevalence Transmission Clinical manifestation and diagnosis Therapeutic treatment of hepatitis B Interferon therapy Lamivudine therapy Thymosin- α Other alternative therapies Hepatitis B viral mutants Hepatitis B surface antigen (pres/s) mutants Hepatitis B DNA polymerase mutants Hepatitis B x gene and related cis-acting region mutants Precore/core gene mutants Quasispecies Aims of the project 50 CHAPTER 2 MATERIALS AND METHODS Materials Biological materials 54 iv

5 Chemicals, reagents and equipment Subjects Methods Genotyping DNA extraction PCR and restriction fragment length polymorphism HBV DNA Quantitation Direct sequencing PCR of core promoter/precore region Purification of PCR product DNA Sequencing HBV variability study Denaturing gradient gel electrophoresis (DGGE) Cloning and sequencing Phylogenetic tree analysis HBeAg expression in HepG 2 cells Mutant constructs preparation Cloning Transient co-transfection HBeAg assay β-galactosidase assay RT-PCR Immunofluorescent staining 71 v

6 Statistical analysis 72 CHAPTER 3 GENOTYPES OF HBV IN SINGAPOREAN CHRONIC HEPATITIS B PATIENTS Introduction Patient Profile and Experimental Design Patients Experimental Design Results Analysis of restriction fragment length polymorphism Phylogenetic analysis of the pre-s/s region Distribution of HBV genotypes in chronic hepatitis B patients Discussion 81 CHAPTER 4 HBV DNA LEVELS AND HEPATITIS B E ANTIGEN SEROCONVERSION Introduction Patient Profile and Experimental Design Patients Experimental Design Results Clinical characteristics of patients Analysis of HBV DNA levels in different genotype patients Dynamic change of HBV DNA levels in chronic hepatitis B patients 88 vi

7 Comparison of HBV DNA levels between groups of chronic hepatitis B patients Discussion 90 CHAPTER 5 RELATIONSHIP BETWEEN HBV MUTATIONS IN THE CORE PROMOTER/PRECORE AND HEPATITIS B E ANTIGEN SEROCONVERSION Introduction Patient Profile and Experimental Design Patients and sera samples Experimental Design Results Clinical characteristics of patients studied Prevalence of the A1762T/G1764A and G1896A mutations Other nucleotide changes in the core promoter region New transcription factor binding sites in the HBV core promoter Discussion Core promoter A1762T/G1764A and precore G1896A mutations Other mutations in the core promoter that involve in transcription bind sites and regulatory regions 104 CHAPTER 6 EVOLUTION OF HBV QUASISPECIES AND HEPATITIS B E SEROCONVERSION Introduction Patient Profile and Experimental Design 110 vii

8 Patients Experimental Design Results Clinical status of the patients in the quasispecies study Optimization of denaturing gradient gel electrophoresis conditions using colony PCR Variability of HBV precore/core gene in chronic hepatitis B patients assessed by denaturing gradient gel electrophoresis Quantification of the genetic variation in HBV precore/core viral quasispecies Genetic divergence - inter time point distance of the HBV 117 precore/core gene Genetic heterogeneity intra time point distance of the 119 precore/core gene Evolution force analysis - dn/ds HBV quasispecies evolution determined by phylogenetic analysis of HBV precore/core gene Longitudinal analysis of HBV precore amino acid sequence HBeAg defective mutants in viral quasispecies in chronic hepatitis B patients Discussion Variability of the HBV precore/core populations determined by DGGE The diversity of HBV precore populations and evolution in vivo Phylogenetic evolution of the HBV quasispecies Relationship between evolution of the HBV precore/core sequences and clinical outcome 142 viii

9 The rate of aa substitutions in HBV precore coding sequence in chronic hepatitis B patients Analysis of HBeAg defective mutants in different groups of chronic hepatitis B patients 145 CHAPTER 7 HEPATITIS B E ANTIGEN EXPRESSION OF THE PRECORE/CORE MUTANTS IN HEPG2 CELLS Introduction Samples and Experimental Design Samples Experimental Design Results HBeAg secretion in the media of transfected HepG 2 cells Expression of HBV precore transcripts in transfected cells Immunofluorescent staining the transfected HepG 2 cells Discussion Mutations that may influence the HBeAg expression in transfected HepG 2 cells HBeAg defective mutants and HBeAg status in chronic hepatitis B patients 161 CHAPTER 8 GENERAL DISCUSSION AND CONCLUSIONS Genotypes and outcomes of chronic hepatitis B infection in Singapore HBV DNA levels change during the course of chronic hepatitis B infection Mutations in the core promoter and precore regions may be associated with HBeAg seroconversion 167 ix

10 Core promoter mutation A1762T/G1764A and precore stop mutation G1896A Other mutations that may affect transcription factor binding and the CP regulatory regions may be associated with HBeAg SC Dynamic change of the precore mutation G1896A in HBV quasispecies during the course of HBV infection Relationship between other precore/core mutations in viral quasispecies and HBeAg SC HBV precore/core quasispecies HBV precore/core sequences exist as quasispecies in chronic hepatitis B patients Evolution of the HBV precore/core quasispecies during the course of HBV infection Conclusions 181 REFERENCE 184 APPENDIX 215 Appendix A: Figure Appendix B: Reagents and equipment 223 Appendix C: Buffers and solutions 229 x

11 LIST OF FIGURES Figure 1. Life cycle of HBV 5 Figure 2. Transcripts and promoters 8 Figure 3. Replication of the HBV gnome 11 Figure 4. Electron micrograph of HBV 14 Figure 5. HBV genome and open reading frames 15 Figure 6. Organization of the HBV core promoter 18 Figure 7. Transcription factor binding sites on the HBV core promoter 20 Figure 8. Schematic model of the HBV and HBs particles 22 Figure 9. Expression of core and e proteins from the precore/core gene 23 Figure 10. Outcome of hepatitis B virus infection in adults 34 Figure 11. RFLP of pre-s analysis 77 Figure 12. Unrooted tree obtained by phylogenetic analysis of the pre-s region 78 Figure 13. Distribution of HBV genotypes in different groups of chronic hepatitis B patients 80 Figure 14. HBV DNA levels at different time points in four groups of chronic hepatitis B patients 88 Figure 15. Nucleotide changes in the core promoter over time in different patients 99 Figure 16. Frequency of different groups of patients with nucleotide changes at different sites in the core promoter over time. 100 Figure 17. Optimization of the DGGE conditions using different ranges of denaturing gradient gel 114 Figure 18. DGGE profiles of the 278-bp fragment in the HBV precore/core region from serial sera samples in different groups of chronic hepatitis B patients 115 xi

12 Figure 19. The inter-time point genetic divergence over time in different groups of patients (Mean ± SE) 118 Figure 20. The intra-time point genetic heterogeneity of DNA sequences over time in different groups of patients (Mean ± SE) 119 Figure 21. Comparison of the inter-time point ratio of dn/ds in different groups of patients 122 Figure 22. Comparison of the intra-time point ratio of dn/ds in different groups of patients 123 Figure 23. Neighbour-joining phylogenetic trees of HBV viral DNA sequences for each patient 127 Figure 24. Average numbers of substitutions per 100 amino acids in deduced precore/core amino acid sequences from different groups of patients 130 Figure 25. Comparison of the frequency of HBV viral sequences with precore mutation G1896A (stop codon mutation at codon 28) and frame shifts over time in each group of patients 134 Figure 26. Standard curve of HBeAg concentration (a) and β-galactosidase activity (b) 150 Figure 27. Alignment of precore amino acid sequences of the mutants in the HBeAg expression study 153 Figure 28. RT PCR analysis of β-actin (a) and the precore/core transcripts for different mutants (b) 155 Figure 29. Immunofluorescent staining of HepG 2 cells transfected with different mutants that were defective in HBeAg secretion 156 Figure 30. Positions where CP mutations were found in chronic hepatitis B patients in this study 170 Figure 31. Figure 31. Mixed wild type and mutated nucleotides at nt in the precore 176 Figure 32. Amino acid changes in deduced HBV precore amino acid sequences of the viral quasispecies 216 xii

13 LIST OF TABLES Table 1. Transcription factors (TFs) required for activation of HBV enhancers 16 and promoters Table 2. Amino acid residues specifying determinants of HBsAg 28 Table 3. Geographical distribution of HBV genotypes and subtypes 29 Table 4. Different mutations in the precore/core gene 46 Table 5. Sequencing gel mixture 60 Table 6. Denaturing gradient gel mixture 61 Table 7. Characteristics of the 71 chronic hepatitis B patients genotyped 76 Table 8. Comparison of Characteristics in chronic hepatitis B patients with different HBV genotypes 79 Table 9. Clinical data of 52 chronic hepatitis B patients in HBV DNA quantitation study 86 Table 10. HBV DNA levels in genotype B and C chronic hepatitis B patients 87 Table 11. Clinical parameters of chronic hepatitis B patients in the direct sequencing of the HBVcore promoter/precore gene 96 Table 12. Frequency of CP mutation A1762T/G1764A and precore mutation G1896A at two time points in different groups of patients 97 Table 13. Characteristics of patients in HBV quasispecies study 112 Table 14. Frequency of clones with aa substitutions in precore sequence (%) 131 Table 15. Normalized HBeAg levels in the media of HepG2 cells transfected with different mutant precore/core genes 152 xiii

14 SUMMARY HBeAg seroconversion is an important clinicopathological event in the natural history of chronic hepatitis B that results in 3 log decrease in HBV DNA, remission of liver disease and improved clinical outcomes. The relation between viral mutations and this event were explored initially using paired serum (before and after) from 33 patients with spontaneous or interferon induced HBeAg seroconversion. Genotype B was associated with higher prevalence rates of seroconversion, and sequencing of the core promoter region was performed to examine the impact of mutations in this region on previously described transcription factor binding sites, as an explanation for the decrease in viral replication and load. Compared to 10 interferon treated non-seroconverters, seroconverters had universally, mutations that affected at least one transcription factor binding site. In addition, precore stop codon mutations (G1896A) and the TA 1762/1764 mutation were seen in pre-seroconversion samples, a surprising finding. This raised the possibility of mixed viral species being present and characterization of these species was performed by PCR, cloning and sequencing of the 639-bp precore/core gene, described as an important immunogenic region. From each serum sample, at least 20 clones were sequenced. The clinical groups were expanded from the 5 patients with spontaneous seroconversion and 5 patients with interferon induced seroconversion to include interferon treated non-responders (5), and non-treated controls (5) who were persistently HBeAg positive. Patients were selected if they had stored serum comprising 4-5 time points over a 5-8 year period, and had only genotype B. Characterization of quasispecies was performed by DGGE and sequencing. DGGE gels demonstrated that only seroconverters had a high proportion of quasispecies with little variation in non- xiv

15 seroconverters. Analysis of sequences from the viral clones showed that there was considerably viral divergence and heterogeneity, most marked in spontaneous seroconverters, and somewhat less so for interferon induced seroconverters but a considerably lower rate of mutations in non-seroconverters. Separately, studies of selected mutations that affected HBeAg secretion was demonstrated by identifying their entrapment in golgi and endoplasmic reticulum using specific antibodies. Taken together, a hypothesis of HBV viral behavior can be postulated. The increase in viral diversity before seroconversion may explain the increase in immune response due to generation of many new viral epitopes to previously tolerised viral antigens. Spontaneous seroconverters had more extreme diversity than interferon induced seroconverters, suggesting that immunomodulator therapy is needed in patients where diversity is insufficient to stimulate the immune system to clear virus by itself. Clearance of immunoreactive strains occurs after seroconversion, with a decrease in viral diversity. Following seroconversion, HBV DNA is 3 log lower, indicating strains which are less replication efficient. Core promoter mutations exist in these strains that affect transcription factor binding sites which may contribute to replication efficiency. Finally, mutations may affect secretion of eag due to entrapment in Golgi or endoplasmic reticulum as an explanation for the absence of HBeAg in the serum when the classic stop codon mutation is absent. Viral mutations of HBV clearly have a highly significant impact on viral behavior and consequently natural history of the disease. xv

16 PUBLICATIONS AND CONFERENCE ABSTRACTS PUBLICATIONS 1. Cheng Y, Lim, SG, Seet BL, Ong C, Tan, TMC, and Peter F. Mutations in the core promoter region following HBeAg seroconversion affect transcription factor binding sites. Under preparing, to be submitted to Hepatology. 2. Cheng Y, Lee LY, Ong C, Seet BL, Kajiji TS, Tan, TMC, Peter F and Lim, SG. Clinical phenotypes of chronic hepatitis B are closely related to HBV quasispecies diversity and heterogeneity. Under preparing, to be submitted to Gastroenterology. ABSTRACTS 1. Cheng, Y, Ong, C, Kausalita, PJ, Seet, BL, Sutedja, DS, Wai, D, Tan, TMC, Lim, SG and Peter, F. Hepatitis B e - seroconversion correlates with clustered changes in the core promoter and enhancer II region of HBV. J. Gastroenterol. Hepatol. 2000; 15 suppl.:j Cheng, Y, Ong, C, Kausalita, PJ, Seet, BL, Sutedja, DS, Wai, D, Tan, TMC, Lim, SG and Peter, F. Hepatitis B e - seroconversion is associated with a three-log drop in viral DNA. J. Gastroenterol. Hepatol. 2000; 15 suppl.:j Peter, F, Cheng, Y, Ong, C, Kausalita, PJ, Seet, BL, Sutedja, DS, Wai, D, Tan, TMC and Lim, SG. No correlation between hepatitis B virus e - seroconversion and hotspot mutations in core promoter and precore sequence of HBV. J. Gastroenterol. Hepatol. 2000; 15 suppl.:j Cheng, Y, Ong, C, Kausalita, PJ, Seet, BL, Sutedja, DS, Wai, D, Tan, TMC, Lim, SG and Peter, F. Hepatitis B e - seroconversion is associated with a three-log drop in viral DNA. Singapore GE Society Annual Scientific Meeting, Oct (oral presentation) xvi

17 ABBREVIATIONS Amino acids Ala A alanine Ile I isoleucine Arg R arginine Leu L leucine Asn N asparagine Lys K lysine Asp D aspartic acid Met M methionine Asx B asparagine or aspartic acid Phe F phenylalanine Cys C cysteine Pro P proline Gln Q glutamine Ser S serine Glu E glutamic acid Thr T threonine Glx Z glutamine or glutamic acid Trp W tryptophan Gly G glycine Tyr Y tyrosine His H histidine Val V valine Others A adenine aa amino acid ALT alanine aminotransferase anti-hbc antibody against hepatitis B core antigen anti-hbe antibody against hepatitis B e antigen anti-hbs antibody against hepatitis B surface antigen APS ammonium peroxide sulphate ARP1 apolipoprotein AI regulatory protein 1 BCP basic core promoter bp base pair C cytosine C/EBP CCAAT/enhancer binding protein cccdna covalently closed circular DNA COUP-TF1 ovalbumin upstream promoter-transcription factor 1 xvii

18 CP CTL DGGE dh 2 O DMEM dn DNA dntp ds EDTA ER FCS FTF g g G H 2 O HBcAg HBeAg HBeAg SC HBsAg HBV HBx HCC HCV HDV HGV HIV HMA core promoter cytotoxic T-lymphocyte denaturing gradient gel electrophoresis distilled water Dulbecco s Modified Eagle s Media the rate of nonsynonymous nucleotide substitution per nonsynonymous site deoxyribonucleic acid 2'-deoxynucleotide 5'-triphosphate datp, dctp, dgtp and dttp rate of synonymous nucleotide substitution per synonymous site ethylene diamine tetra-acetic acid endoplasmic reticulum fetal calf serum fetal protein transcription factor gravity in centrifuge force gram in weight Guanine water hepatitis B core antigen hepatitis B e antigen hepatitis B e antigen seroconversion hepatitis B surface antigen hepatitis B virus hepatitis B x protein hepatocellular carcinoma hepatitis C virus hepatitis D virus hepatitis G virus human immunodeficiency virus heteroduplex mobility analysis xviii

19 HNF1 hepatocyte nuclear factor 1 HNF3 hepatocyte nuclear factor 3 HNF4 hepatocyte nuclear factor 4 IFN interferon IPTG isopropylthio-β-d-galactoside kb kilo bases kda kilo Dalton LEF liver enriched factor LHBs large hepatitis B surface protein M molar mg milligram MgCl 2 magnesium chloride MHBs middle hepatitis B surface protein MHC major histocompatibility complex min minute ml milliliter mm millimolar mrna messenger RNA NaCl sodium chloride NaOAc sodium acetate NaOH sodium hydroxide NCBI national center for biotechnology information ng nanogram nm nanometer NRE negative regulatory element OD optical density ORF open reading frame PCR polymerase chain reaction PFA paraformaldehyde pgrna pregenomic RNA pmol picomole xix

20 PPAR peroxisome proliferator-activated receptor pre-c precore RFLP restriction fragment length polymorphisms RNA ribonucleic acid RT-PCR reverse transcriptional polymerase chain reaction RXR retinoid X receptor SC seroconversion SdH 2 O sterile distilled water SDS sodium deodecyl sulphate SE standard error of mean SHBs small hepatitis B surface protein Sp1 stimulating protein 1 SSCP single-strand conformation polymorphism T thymine TAE tris-acetate-edta TBE tris-borate-edta TBP TATA box binding protein TE tris-edta TEMED N,N,N,N-tetramethyl-ethylenediamine Tris tris (hydromethyl) methylamine URR upper regulatory region UV ultra violet V volts X-gal 5-bromo-4-chloro-3-indolyl- β-d-galactoside µg microgram µl microliter µm micromolar xx