THE ROLE OF DEFECTIVE HEPATITIS B VIRUS IN WILD- TYPE HEPATITIS B VIRUS REPLICATION
MANISH KANDPAL
KUSUMA SCHOOL OF BIOLOGICAL SCIENCES INDIAN INSTITUTE OF TECHNOLOGY DELHI
FEBRUARY 2017
©Indian Institute of Technology Delhi (IITD), New Delhi, 2017
THE ROLE OF DEFECTIVE HEPATITIS B VIRUS IN WILD- TYPE HEPATITIS B VIRUS REPLICATION
by
MANISH KANDPAL
Kusuma School of Biological Sciences
Submitted
in fulfilment of the requirements of the degree of Doctor of Philosophy
to the
Indian Institute of Technology Delhi
FEBRUARY 2017
Dedicated to my beloved family.
My parents and my sister
for their unconditional love…
CERTIFICATE
i
This is to certify that the thesis titled “The role of defective hepatitis B virus in
wild-type hepatitis B virus replication”, being submitted by Mr. Manish Kandpal to the Indian Institute of Technology Delhi, for the award of the degree of “Doctor of Philosophy” is a record of the bonafide research carried out by him, which has been prepared under my supervision and guidance in conformity with rules and regulation of the Indian Institute of Technology Delhi, India. The results prescribed in it have not been submitted in part or full to any other University or Institute for the award of any Degree/Diploma.
Date: Dr. Vivekanandan Perumal New Delhi Associate Professor Kusuma School of Biological Sciences Indian Institute of Technology Delhi New Delhi-110016, India
ACKNOWLEDGEMENTS
ii
First and foremost I would like to thank my Ph.D. advisor Dr. Vivekanandan Perumal. I would like to appreciate his contribution in terms of time, ideas and funding to make my Ph.D. a great learning experience. He has helped me sail through tough times and has motivated me to pursue a career in research. Being one of the first Ph.D. students enrolled in his lab, I have not only gained scientific exposure but have also experienced and understood the difficulties that arise in establishing a functional lab. I sincerely thank him for his contribution in designing of experiments, data analysis and preparation of manuscripts.
I would like to thank my student research committee (SRC) members: Prof.
Chinmoy Shankar Dey, Dr. Archana Chugh and Dr. Nivedita K. Gohil (Center for Biomedical Engineering) for their valuable suggestions and timely guidance to help me improve my research work.
I would also like to thank the co-ordinator of the Kusuma School of Biological Sciences Prof. James Gomes and all the faculty members (Prof. Chinmoy Shankar Dey, Prof. Tapan Kumar Chaudhuri, Prof. Aditya Mittal, Prof. B. Jayaram, Prof. S.E. Hasnain, Dr. Bishwajit Kundu, Dr. Archana Chugh, Dr. Manidipa Banerjee and Dr. Ashok Kumar Patel) for their constant support, encouragement and making all the facilities accessible round the clock required for conducting the experiments.
I want to thank our departmental storekeeper Mr. Inderjeet and staff members Ms. Mini, Ms. Pushplata, Ms. Punita, Mr. Mukesh and Mr. Vijay Pal for smooth processing of administrative work.
I would like to thank Dr. Vimarsh Raina (Medanta, The Medicity, Gurgaon) for providing us the clinical samples to begin my work.
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I would like to thank Department of Biotechnology (DBT), Government of India and Kusuma Trust, UK for funding my project. I would like to thank IIT Delhi for providing me Junior and Senior Research Fellowship during my Ph.D. and funding to attend an international conference in China.
I would also like to appreciate the contribution of my lab members Banhi, Jasmine, Dr. Mohita and new entrants during recent years Shivani, Nandhini, Kiruthika, Dr. Jayant and Dr. Mishi who helped in building a core research group. I would like to specially thank Banhi for helping me in my experiments by giving new ideas and in experimental troubleshooting. She has been a constant support and encouragement especially during last two years of my Ph.D. I would also like to thank Ashutosh Shukla, Ankit, Saumya,Aakash and Deepthi for their help and assistance in carrying out my work.
I would like to acknowledge the support and motivation that my friends have provided me throughout the tenure of my Ph.D. I am especially thankful to my friends Dr. Aastha and Dr. Amita who have supported me during the most difficult times during Ph.D. and always encouraged me. Besides that, their inputs to some experiments were quite helpful and are invaluable. I am thankful to my friend Anuj at IIT Madras who has helped me immensely throughout my Ph.D. with experimental troubleshooting and providing new ideas. We have been together for almost 8 years since my master’s programme at Anna University and we share a strong bond. I also want to thank my friends Dr. Sanjeev who graduated from IIT Delhi last year and Arghya (currently a Ph.D. student at CRDT, IIT Delhi) for whatever possible help they could do to smoothen my Ph.D. and also for all the joyful sessions with them.
Last but not the least I want to thank my family for all their love, encouragement and support without which it would not have been possible for me to
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reach up to this level. They have helped me in setting my career goals and achieving them. Though I have always stayed away from them since my childhood days, their sacrifice and contribution cannot be described in words. I am thankful to my sister Vidisha who was always there to share my problems and always motivated me despite being 5 years younger to me. Finally, I am thankful to the AlmightyGod for providing me strength to accomplish my Ph.D.
MANISH KANDPAL
ABSTRACT
v
Hepatitis B virus is a small DNA virus that can cause both acute and chronic infections. According to the reports of world health organization, an estimated 2 billion people are exposed to HBV infection worldwide, of which 250 million people remain chronically infected with HBV. Chronic infection with HBV is a major risk factor for hepatocellular carcinoma.
Hepatitis B virus proteins are typically produced from unspliced HBV messenger RNAs (mRNA). In addition, the HBV pregenomic RNA (pgRNA) undergoes splicing at various sites leading to the production of spliced pgRNAs. The spliced pgRNAs can be reverse transcribed by viral polymerase to its corresponding HBV DNA that can be packaged in the core protein and the packaged nucleocapsids are enveloped and secreted as defective HBV particles (dHBV). The splice variants of pgRNA and their corresponding dHBV particles have been detected in patients with acute/chronic HBV infection as well as in HBV cell culture. Among the HBV splice variants, the singly-spliced pgRNA that is produced as a result of splicing between the nucleotides 2447 and 489 in the HBV pgRNA is the most predominant form. The presence of splice sites in HBV genome is necessary for the production of these spliced RNA; a point mutation at either of the splice site inhibits pgRNA splicing and subsequently abrogates the generation of dHBV particles. Another unique characteristic of the singly spliced pgRNA is that it encodes for a novel fusion protein, the HBV-generated splice protein (HBSP). Higher dHBV/wtHBV ratios in chronic HBV patients are linked to severe liver necrosis and fibrosis. Furthermore, clinical studies have linked the presence of dHBV encoded HBSP to high virus loads and liver damage; however the functional role of dHBV particles and dHBV encoded HBSP in HBV replication is not known.
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In this study, we analyzed the splice donor and splice acceptor sites (corresponding to singly-spliced pgRNA) in the HBV genome from different HBV genotypes (A-H). We found that the splice donor/acceptor sites essential for the formation of dHBV particles corresponding to singly-spliced pgRNA are conserved across HBV genotypes (A-H). We report a novel method to create dHBV constructs from corresponding wild-type HBV constructs. Using the cell culture model we developed, the replication characteristics of dHBV constructs were assessed and compared with that of corresponding wild-type constructs in cell culture. Interestingly, dHBV constructs have higher pgRNA levels, transcription efficiency, HBeAg levels and intracellular HBcAg levels as compared to that of corresponding wild-type HBV constructs. Furthermore, we demonstrate that the dHBV encoded HBSP selectively up regulates HBV preS2/S promoter activity and increases the secretion of hepatitis B surface antigen (HBsAg) and virions in cell culture. To our knowledge, this is the first report to demonstrate the role of dHBV particles in the replication of wild-type HBV.
Our findings highlight previously unrecognized fundamental molecular characteristics of dHBV genomes and their role in HBV replication.
lkjka”k
vii
gsisVkbfVl ch fo’kk.kq ¼,p0ch0oh0½ ,d lw{e Mh0,u0,0 fo’kk.kq gS tks rhoz rFkk nh?kZdkfyd nksuksa ladze.k iSnk dj ldrk gSA fo”o LokLF; laxBu dh fjiksVZ ds vuqlkj iwjs fo”o esa yxHkx nks vjc yksx ,p0ch0oh0 ls xzflr gSaA ftuesa ls 2500 yk[k yksx nh?kZdkfyd ladze.k ls xzflr gSaA fgisVkslsyqyj dklhZuksek dk ,d izeq[k dkjd ,p0ch0oh0 fo’kk.kq dk nh?kZdkfyd ladze.k gSA
gsisVkbfVl ch fo’kk.kq ¼,p0ch0oh0½ ds izksVhu dh mRifRr fof”k’V :i ls vuLiykbLM ,p0ch0oh0 eSlsatj vkj0,u0,0 ls gksrh gSA blds lkFk&lkFk ,p0ch0oh0 izhthuksfed vkj0,u0,0 fofHkUu fljksa ij LIykbflax ls xqtjrk gSA ftuls LiykbLM izhthuksfed vkj0,u0,0 dk mRiknu gksrk gSA LiykbLM izhthuksfed vkj0,u0,0 izfrykse ys[ku ds }kjk iksfyejst izksVhu dh lgk;rk ls ,p0ch0oh0 Mh0,u0,0 esa cnyrs gSa tks fd dksj izksVhu esa iSad gks ldrs gSaA iSadsTM U;wdfy;ksdSilkbM ifjxzfgr dj ysus ij ,p0ch0oh0 ds nks’k iw.kZ d.k ¼fM0,p0ch0oh0½ ds :i esa lzkfor dj fn;s tkrs gSaA izhthuksfed vkj0,u0,0 ds fofHkUu :iksa vkSj muds le:ih nks’k iw.k ,p0ch0oh0 ¼fM0,p0ch0oh0½ d.kksa dks rhoz nh?kZdkfyd ,p0ch0oh0 ladzfer jksfx;ksa ds lkFk&lkFk ,p0ch0oh0 dksf”kdk lao/kZu esa ik;k x;k gSA ,p0ch0oh0 LiykbLM vkj0,u0,0 ds fofHkUu :iksa esa flaxyh LiykbLM izhthuksfed vkj0,u0,0 izeq[k gSaA ftldh mRifRr U;wfD;ksVkbM 2447 vkSj 489 ds chp Liykbflax ds ifj.kkeLo:i gksrh gSA ,p0ch0oh0 thukse esa Liykbl LFky Liykbl vkj0,u0,0 ds mRiknu ds fy;s vfuok;Z gSA dksbZ ,d LiykbLM LFky ij fcUnq mRifjorZu gksus ls izhthuksfed vkj0,u0,0 Liykbl esa vojks/ku gksrk gS vkSj ;g fM0,p0ch0oh0 d.kksa dh mRifRr dks jksdrk gSA flaxyh LiykbLM izhthuksfed vkj0,u0,0 dh ,d vkSj fo”ks’krk ;g gS fd ;g uohu izksVhu ¼,p0ch0oh0 mRikfnr Liykbl izksVhu½ ds fy;s dksM djrk gSA fM0,p0ch0oh0∕,p0ch0oh0 ds mPp vuqikr dks nh?kZdkfyd ,p0ch0oh0 jksfx;ksa esa yhoj ifjxyu vkSj rarqe;rk fodkjksa ls tqM+k ekuk tkrk gSA blds vfrfjDr uSnkfud v/;;u Hkh fM0,p0ch0oh0 mRikfnr ,p0ch0,l0ih0 dh mifLFkfr dks mPp fo’kk.kq Lrj vkSj ;d`r dh {kfr esa tksM+rk gSz] fQj Hkh fM0,p0ch0oh0 d.kksa rFkk fM0,p0ch0oh0 mRikfnr ,p0ch0,l0ih0 dh ,p0ch0oh0 izfrd`fr esa dk;kZRed Hkwfedk dk Kku ugha gSA
bl v/;;u esa geus nkrk rFkk xzkgh LFky ¼flaxyh LiykbLM½ dks izhthuksfed vkj0,u0,0 fofHkUu ,p0ch0oh0 thuksVkbi ls ,p0ch0oh0 thukse esa fo”ys’k.k fd;kA geus ik;k fd nkrk rFkk xzkgh blh flaxyh LiykbLM dks izhthuksfed vkj0,u0,0 d.kksa ds fuekZ.k ds fy;s vko”;d fljs ,p0ch0oh0 thuksVkbi ¼,−,p½ ij lajf{kr dj jgs gSaA
ge okbYM Vkbi ,p0ch0oh0 ls fM0,p0ch0oh0 ds fuekZ.k dh ,d uohu i)fr izLrqr djrs gSaA dksf”kdk lao/kZu ds iz;ksx ls geus fM0,p0ch0oh0 ds fuekZ.k rFkk fodkl dh fo”ks’krkvksa dk vkadyu fd;k rFkk dksf”kd lao/kZu esa blh izdkj ds okbYM fuekZ.kksa ds lkFk rqyuk dhA fnypLi ckr ;g gS fd fM0,p0ch0oh0 dk izhthuksfed vkj0,u0,0
viii
Lrj] izfrys[ku {kerk] ,pch^b* izksVhu Lrj rFkk vr%dks”kdh; gsisVkbfVl chs dksj izksVhu Lrj okbYM Vkbi ,p0ch0oh0 ds lkis{k vf/kd ik;k x;kA blds vykok ;g iznf”kZr gqvk fd fM0,p0ch0oh0 mRikfnr ,p0ch0,l0ih0 pqfuUnk ,p0ch0oh0 izh ,l2∕,l izeksVj dh fdz;kfof/k dks fu;af=r djrk gS vkSj fgisVkbfVl ch lrgh izksVhu rFkk fgisVkbfVl ch dksf”kdk lao/kZu ds lzko dks c<+krk gSA gekjh tkudkjh esa okbYM Vkbi ,p0ch0oh0 dh izfrd`fr esa fM0,p0ch0oh0 d.kksa dh Hkwfedk dk ;g izn”kZu dk igyk ekSdk gSA gekjk fu’d’kZ fM0,p0ch0oh0 thukse dh ekSfyd vk.kfod fo”ks’krkvksa rFkk ,p0ch0oh0 izfrd`fr esa mudh Hkwfedk ij izdk”k Mkyrk gSA
TABLE OF CONTENTS
ix
Title Page No.
Certificate i
Acknowledgements ii
Abstract v
Table of Contents ix
List of Figures xii
List of Tables xv
List of Abbreviations xvi
Chapter 1. Review of literature and Aims 1-31
1.1. Introduction 1
1.2. Hepatitis B virus taxonomy 2
1.3. Classification of hepatitis B virus 2
1.4. Hepatitis B virus genome structure and organization 3
1.5. Hepatitis B virus replication 4
1.6. Hepatitis B virus proteins 6
1.7. Pathogenesis of hepatitis B virus infection 9 1.8. Clinical course of hepatitis B virus infection 11
1.9. Hepatitis B virus epidemiology 14
1.10. Hepatitis B virus transmission 15
1.11. Diagnosis of hepatitis B virus infection 15
1.12. Treatment of hepatitis B virus infection 17
1.13. Hepatitis B virus vaccine 22
1.14. Epidemiology of hepatitis B virus infection in India 23
1.15. Occult hepatitis B virus infection 24
x
1.16. Defective hepatitis B virus particles 26
1.17. Aims 31
Chapter 2. Designing a cell culture model to study the replication of defective HBV corresponding to singly spliced pregenomic RNA
32-57
2.1. Introduction 32
2.2. Materials and methods 38
2.3. Results and discussion 47
2.4. Conclusion 57
Chapter 3. To assess the replication characteristics of defective HBV compared to wild-type HBV
58-75
3.1. Introduction 58
3.2. Materials and methods 61
3.3. Results and discussion 65
3.4. Conclusion 75
Chapter 4. To investigate the role of HBSP (encoded by defective HBV) in the replication of wild-type HBV
76-102
4.1. Introduction 76
4.2. Materials and methods 79
4.3. Results 87
4.4 Discussion 101
4.5. Conclusion 105
Summary 106-107
References 108-138
Appendix 139-143
List of Publications and conferences 144-145
xi
Author’s Resume 146
LIST OF FIGURES
xii
Number Title Page No.
Figure 1.1 A schematic representation of the HBV genome 3 Figure 1.2 Overview of mechanisms leading to occult hepatitis B
virus (HBV) infection
26
Figure 2.1 Splice variants of HBV pregenomic RNA detected in chronic HBV patients
35
Figure 2.2 Electropherograms 41
Figure 2.3 Schematic diagram describing the creation of dHBV construct
42
Figure 2.4 Nucleotide sequence alignment of splice donor/
acceptor sites
48
Figure 2.5 Agarose gel electrophoresis to analyze the full length HBV DNA amplified using PCR
49
Figure 2.6 Agarose gel electrophoresis for the analysis of screening of wild type HBV clones
50
Figure 2.7 Agarose gel electrophoresis to analyze PCR amplified dHBV DNA
51
Figure 2.8 Agarose gel electrophoresis for analysis of dHBV (PCR amplified dHBV genome using primers D1 and D2) clones
52
Figure 2.9 Agarose gel electrophoresis to analyze the PCR amplification of dHBV DNA using primers P1 and P2
53
Figure 2.10 Agarose gel electrophoresis for the analysis of dHBV 54
xiii
(PCR amplified dHBV genome using primers P1 and P2) clones
Figure 2.11 Schematic diagram showing the primer and probe binding regions for HBV cccDNA real time PCR assay
55
Figure 2.12 Standard curves used for the absolute quantitation 56 Figure 3.1 Replication characteristics of the dHBV genome as
compared to the corresponding wild-type HBV genome.
67
Figure 3.2 Estimation of HBV proteins in cells transfected with the dHBV and the wild-type HBV constructs.
69
Figure 3.3 Agarose gel electrophoresis to analyze the PCR amplified pkex-1+2 mutant HBV DNA
70
Figure 3.4 Agarose gel electrophoresis for the analysis of screening of pkex1+2 (replication incompetent) HBV DNA clones
71
Figure 3.5 Validation of a novel assay for the estimation of hepatitis B virions in cell culture
72
Figure 3.6 Quantitation of hepatitis B virions in serum samples from HBV patients and in cell culture supernatants
73
Figure 4.1 The amino acid sequence alignment of HBSP across HBV genotypes (A-H)
89
Figure 4.2 Replication characteristics of wild-type and splice mutant HBV constructs
87
Figure 4.3 Estimation of secreted HBsAg in cells transfected with the wild-type and splice mutant constructs
88
xiv
Figure 4.4 Estimation of secreted HBsAg in Huh7 cells co- transfected with splice mutant and dHBV constructs
90
Figure 4.5 Western blot analysis of the HBSP expression in Huh7 cells
91
Figure 4.6 Estimation of secreted HBsAg in cells co-transfected with the splice mutant and the HBSP constructs
92
Figure 4.7 Quantitation of extracellular HBeAg in cells co- transfected with the splice mutant and the HBSP-N constructs
93
Figure 4.8 Replication characteristics of HBV DNA in cells co- transfected with the splice mutant and HBSP-N construct
95
Figure 4.9 Southern blot to analyze HBV cccDNA 95 Figure 4.10 Estimation of secreted virions in cells co-transfected
with the splice mutant and HBSP-N constructs
96
Figure 4.11 Estimation of HBV surface transcript levels and secreted HBsAg in cells co-transfected with the surface construct and the HBSP-N construct.
97
Figure 4.12 Luciferase assay to analyze the effect of HBSP-N on HBV promoters.
99
Figure 4.13 Luciferase assay to analyze the effect of HBSP-S on HBV preS2/S promoter
100
LIST OF TABLES
xv
Number Title Page No.
Table 1 The probe and primer sequences used in real-time PCR 46 Table 2 The primer sequences used for PCR amplification of desired
region in the HBV genome
84
ABBREVIATIONS & SYMBOLS
xvi
ALT Alanine transaminase
β-globin Beta-globin
cccDNA Covalently closed circular DNA CD4 Cluster of differentiation 4 CD8 Cluster of differentiation 8
cDNA Complementary DNA
CHB Chronic hepatitis B
CTSB Cathepsin B
dHBV Defective hepatitis B virus
DNA Deoxyribonucleic acid
DR Direct repeat
ELISA Enzyme linked immunosorbent assay
ER Endoplasmic reticulum
GAPDH Glyceraldehyde 3-phosphate dehydrogenase HBcAg Hepatitis B core antigen
HBeAg Hepatitis B e antigen HBsAg Hepatitis B suface antigen
HBV Hepatitis B virus
HCC Hepatocellular carcinoma
HCV Hepatitis C virus
HIV Human immunodeficiency virus
Hsp Heat shock protein
IFN Interferon
xvii
IgG Immunoglobulin G
IgM Immunoglobulin M
mRNA Messenger RNA
NCBI National Center for Biotechnology Information NTCP Sodium taurocholate cotransporting polypeptide OBI Occult hepatitis B virus infection
ORF Open reading frame
PCR Polymerase chain reaction
PEG Polyethylene glycol
pgRNA pre-genomic RNA
RC-DNA Relaxed circular DNA
RIPA Radio immune precipitation assay
RNA Ribonucleic acid
wt Wild type
IU International Unit
kb Kilobases
KDa Kilodalton
µM Micromolar
mL Millilitre
mM Millimolar
nm Nanometer
OD Optical density
rpm Rotation per minute
