Influence of hepatitis B virus surface protein variants associated with antiviral resistance on viral

127 Pages·2014·14.22 MB·English

by  Mohr, Christina

Angefertigt am
Institut für Medizinische Virologie
Nationales Referenzzentrum für Hepatitis-B- und D-Viren
der Justus-Liebig-Universität Gießen

Influence of Hepatitis B Virus Surface Protein Variants
Associated with Antiviral Resistance on Viral Assembly
and Secretion of Hepatitis B and Hepatitis D Viruses

Einfluss von Hepatitis B Virus Oberflächenproteinvarianten, die assoziiert sind mit antiviraler
Resistenz, auf das virale Assembly und die Sekretion von Hepatitis B und Hepatitis D Viren

Inauguraldissertation
zur Erlangung des akademischen Grades
d o c t o r r e r u m n a t u r a l i u m
(Dr. rer. nat.)
des Fachbereiches 08-Biologie und Chemie
der Justus-Liebig Universität Gießen

vorgelegt von
Christina Mohr MSc.-Biol.

Gießen 2014
Dekan: Prof. Dr. Holger Zorn

Gutachter: Prof. Dr. Michael Martin
PD Dr. Dieter Glebe
Pour tous les petits héros,
qu`ils attendent dans l'ombre le retour du soleil
Zaz; Recto Verso, 2013

Für all die kleinen Helden,
die im Schatten darauf warten in die Sonne zurück zu kehren
Index
Index

LIST OF ABBREVIATIONS ................................................................................................................ I
ABSTRACT ......................................................................................................................................... IV
ZUSAMMENFASSUNG ...................................................................................................................... V
1. INTRODUCTION ......................................................................................................................... 1
1.1. Viral Hepatitis ............................................................................................................................ 1
1.1.1. Infection with the Hepatitis B Virus (HBV) ........................................................................ 1
1.1.2. HBV/HDV Co- and Super-Infection ................................................................................... 1
1.1.3. Seroepidemiology and Transmission of the Hepatitis B and D Virus ................................. 2
1.1.4. Prevention of an HBV- and HDV-Infection ........................................................................ 3
1.1.5. Therapy of Chronic HBV and HDV-Infection .................................................................... 3
1.2. Hepatitis B Virus (HBV) ........................................................................................................... 4
1.2.1. Classification ....................................................................................................................... 4
1.2.2. Viral Properties .................................................................................................................... 5
1.2.2.1. Morphology and Structure ............................................................................................... 5
1.2.2.2. Genomic Structure and Organization .............................................................................. 6
1.2.2.3. Viral Surface Proteins...................................................................................................... 8
1.2.3. Viral Life Cycle and Replication ....................................................................................... 10
1.3. Hepatitis Delta Virus (HDV) .................................................................................................. 12
1.3.1. Classification ..................................................................................................................... 12
1.3.2. Morphology and Genomic Structure ................................................................................. 13
1.3.3. Viral Life Cycle ................................................................................................................. 14
1.4. Model Systems for HBV and HDV ........................................................................................ 16
1.5. Aim of the Work ...................................................................................................................... 18
2. MATERIALS AND METHODS ................................................................................................ 19
2.1. Materials................................................................................................................................... 19
2.1.1. Antibiotics ......................................................................................................................... 19
2.1.2. Antibodies and Nuclear Staining ....................................................................................... 19
2.1.2.1. Primary Antibodies ........................................................................................................ 19
2.1.2.2. Secondary Antibodies .................................................................................................... 19
2.1.2.3. Nuclear Staining ............................................................................................................ 20
2.1.3. Peptide ............................................................................................................................... 20
2.1.4. Antivirals ........................................................................................................................... 20
2.1.5. Buffer, Media and Solutions .............................................................................................. 20
2.1.5.1. Buffers ........................................................................................................................... 20
2.1.5.2. Media ............................................................................................................................. 21
Index
2.1.5.3. Solutions ........................................................................................................................ 22
2.1.6. Commercial Kits, Enzymes and Competent Bacteria ....................................................... 22
2.1.7. Cell Lines .......................................................................................................................... 23
2.1.8. Viruses Derived from Serum/Plasma of Chronically Infected Individuals ....................... 24
2.1.9. Plasmids ............................................................................................................................. 24
2.1.9.1. Standard Plasmids ......................................................................................................... 24
2.1.9.2. HBV-Expression Plasmids for HBV Phenotypic Assay ............................................... 24
2.1.9.3. Plasmids for Analyzing HBV Variants Harbouring Stopmutants within the S-ORF .... 25
2.1.9.4. Plasmids Encoding HBV Surface Proteins for Generating Recombinant HDV in Cell
Culture ........................................................................................................................... 25
2.1.10. PCR, Primer, Probes and Mastermixes ............................................................................. 25
2.1.10.1. Primer and TaqMan Probes for Quantitative Polymerase Chain Reaction (qPCR) .. 25
2.1.10.2. Primer for Generating an HDV-RNA Standard ........................................................ 26
2.1.10.3. Sequencing Primer for HDV-RNA-Standard ............................................................ 26
2.1.10.4. Mastermixes for Hot Start and Gradient PCR ........................................................... 26
2.1.10.5. qPCR Mastermixes .................................................................................................... 26
2.1.11. Equipment, Consumable Supplies and Software ............................................................... 27
2.2. Methods .................................................................................................................................... 29
2.2.1. Preparation of an RNA-Standard in vitro for HDV qPCR ................................................ 29
2.2.1.1. Amplification of one HDV Genome out of the Plasmid pSVLD3 ................................ 29
2.2.1.2. Isolation and Purification of the PCR Templates .......................................................... 29
2.2.1.3. Synthesis of RNA in vitro and Phenol/Chloroform Extraction ..................................... 30
2.2.1.4. Formaldehyde Gel ......................................................................................................... 31
2.2.1.5 Calculation of Genome Equivalent................................................................................ 31
2.2.2. Preparation of Competent Bacteria for Transformation (″chemical method″) .................. 31
2.2.3. Transformation .................................................................................................................. 32
2.2.3.1. Transformation of Plasmid-DNA into E.coli ................................................................ 32
2.2.3.2. Plasmid-Preparation with the Plasmid-Plus-Midi-Kit (Qiagen) .................................... 32
2.2.4. Cultivation of Immortalized Cell Lines ............................................................................. 32
2.2.4.1. Freezing and Thawing of Eukaryotic Cell Lines ........................................................... 32
2.2.4.2. Passaging of Immortalized Cell Lines ........................................................................... 33
2.2.5. Phenotypic Resistance Assay for Characterization of Patient Isolates .............................. 33
2.2.5.1. Transfection ................................................................................................................... 33
2.2.5.2. Phenotypic Assay .......................................................................................................... 34
2.2.5.3. Calculation of Transfection-Efficiency by Secreted Alkaline Phosphatase- (SEAP-)
Detection Assay ............................................................................................................. 35
2.2.5.4. Statistical Analysis of the Data from Phenotypic Resistance Assay in vitro ................. 35
2.2.6. Cell Viability Test with WST-1 (Roche) ........................................................................... 36
2.2.7. Isolation of Viral Genomes from Supernatant, Plasma or Cell Lysates ............................ 37
2.2.7.1. Isolation of Intracellular Viral RNA and DNA ............................................................. 37
2.2.7.2. Manual Isolation of Viral RNA and DNA in the Supernatant or in Serum ................... 37
2.2.7.3. Automated isolation of viral DNA in the Supernatant with Freedom EVO .................. 37
2.2.8. qPCR ................................................................................................................................. 38
2.2.8.1. SYBR-Green Based qPCR ............................................................................................ 38
2.2.8.2. TaqMan Probe Based qPCRs ........................................................................................ 38
2.2.9. Preparation of a Sucrose Gradient for Isolation and Purification of HBV and HDV ........ 39
2.2.10. Preparation of a Linear Caesium-Chloride (CsCl)-Gradient ............................................. 40
2.2.11. Isolation and Cultivation of Primary Tupaia Hepatocytes (PTHs) ................................... 40
Index
2.2.12. Infection of PTHs with HBV and HDV ............................................................................ 41
2.2.13. ELISA against Secreted HBV Surface Proteins ................................................................ 41
2.2.14. UREA-Assay ..................................................................................................................... 42
2.2.15. Staining for Immunofluorescence ..................................................................................... 42
3. RESULTS ..................................................................................................................................... 43
3.1. Phenotypic Characterization of Isolates from Chronically Infected HBV-Patients.......... 43
3.2. Characterization of Known HBsAg-Mutations During Antiviral Therapy of Chronic
Hepatitis B ................................................................................................................................ 45
3.2.1. Secretion of Enveloped Virions and Subviral Particles ..................................................... 45
3.2.2. Immunohistochemical Analysis of HBV Surface Protein Variants .................................. 48
3.2.3. Comparison of Sedimentation Behavior for Purified HBV with Cell Culture Derived HBV
in the CsCl-Gradient .......................................................................................................... 53
3.2.4. Analysis of a LHBs Deficient HBV Mutant Using the CsCl-Gradient ............................. 54
3.2.5. Analysis of HBV Variants with Mutations at Position 172 into the SHBs Using the CsCl-
Gradient ............................................................................................................................. 55
3.2.6. Analysis of HBV Variants with Mutations at Position 196 into the SHBs Using the CsCl-
Gradient ............................................................................................................................. 56
3.3. Establishment of a Transfection and Infection System for HDV Pseudo-Particles .......... 57
3.3.1. Production and Establishment of an HDV RNA-standard for RT-qPCR .......................... 57
3.4. Characterization of HDV from Human Plasma ................................................................... 60
3.4.1. Characterization of HDV Derived from Human Plasma in a Linear CsCl-Gradient ........ 60
3.4.2. Purification of Plasma-Derived HDV with a Continuous Sucrose Gradient ..................... 60
3.4.3. Infection of PTHs with Purified HDV Derived from Human Plasma ............................... 61
3.4.4. Analysis of the Kinetic of Infectivity for HBV and HDV on PTH ................................... 62
3.4.5. HBV/HDV Co- and Super-Infection of PTH .................................................................... 63
3.5. Characterization of Recombinant HDV Pseudo-Particles from Cell Culture ................... 65
3.5.1. Production of HDV Pseudo-Particles in Cell Culture with Different Variants of HBV
Surface Proteins ................................................................................................................. 65
3.5.2. Infection of PTHs with Recombinant HDV Pseudo-Particles ........................................... 66
3.5.3. Neutralization of Infection of Cell Culture-Derived and Plasma-Derived HDV Using
Monoclonal Antibodies ..................................................................................................... 67
3.6. Production of Recombinant HDV with Mutations in the HBV Surface Proteins ............. 68
4. DISCUSSION ............................................................................................................................... 69
4.1. Phenotypic Resistance Profile of Special Patient Isolates During Antiviral Therapy ....... 69
4.2. Influence of Mutations within the HBV S-ORF that Arise During Antiviral Therapy on
the Viral Life Cycle of HBV ................................................................................................... 71
4.3. Establishment of an RNA Standard for HDV RT-qPCR .................................................... 76
Index
4.4. Characterization of HDV Derived from Human Plasma of a Chronically Infected
Patient ....................................................................................................................................... 77
4.5. Infection of PTH with Purified HDV ..................................................................................... 77
4.6. Production and Detection of Recombinant HDV Enveloped with Different Combinations
of HBsAg .................................................................................................................................. 79
4.7. Infection of PTH with Recombinant HDV in Comparison with Purified HDV ................ 81
4.8. Influence of Mutations within the HBV S-ORF that Arise During Antiviral Therapy on
the Viral Life Cycle of the Hepatitis D Virus ........................................................................ 82
5. PERSPECTIVES ......................................................................................................................... 85
6. REFERENCES ............................................................................................................................ 86
7. LIST OF OF FIGURES ............................................................................................................ 102
8. LIST OF TABLES ..................................................................................................................... 103
9. APPENDIX................................................................................................................................. 104
10. PUBLICATIONS ....................................................................................................................... 113
11. DANKSAGUNG ........................................................................................................................ 114
12. ERKLÄRUNG ........................................................................................................................... 115
List of abbreviations
List of abbreviations
A EtOH ethanol
ETV entecavir
aa amino acid
F
ADAR-1 double-stranded RNA specific adenosine
deaminase acting on RNA-1
f.c. final concentration
ADV adefovir
FCS fetal calf serum
ANOVA analysis of variants

G
B
g gram
bp base pair
GE genome equivalent

GRE glucocorticoid-responsive element
C

°C celsius
H
cDNA complementary DNA
h hours
cccDNA covalently closed circular DNA
HBcAg hepatitis B virus core antigen
CsCl cesium chloride
HBeAg hepatitis B e antigen
C-terminus carboxy terminus
HBsAg hepatitis B surface antigen

HBSS Hank`s balanced salt solution
D
HBx hepatitis B X protein
Da Dalton
HBV hepatitis B virus
DHBV duck hepatitis B virus
HCC hepatocellular carcinoma
DMEM Dulbecco`s Modified Eagle Medium
HDV Hepatitis Delta virus
DMSO dimethyl sulfoxide
HDAg Hepatitis Delta antigen
DNA Deoxyribonucleic acid
hsp heat-shock-protein
DR1/2 direct repeats
HSPGs heparan-sulfate proteoglycans
ds DNA double stranded DNA

I
E
IC inhibitory concentration
E.coli Escherichia coli
IFN-α interferon-α
EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol tetraacetic acid
L
ELISA enzyme linked immunosorbent assay
l litre
ER endoplasmic reticulum
LB-medium lysogeny broth medium
EtBr ethidium bromide
LHBs large hepatitis B virus surface antigen
I
List of abbreviations
L-HDAg large hepatitis Delta virus antigen PEG polyethylene glycol
LMV lamivudine PFA paraform aldehyde
pgRNA pregenomicRNA
M PHH primary human hepatocytes
µ micro p.i. post infection
µl micro litre pol polymerase
m milli/meter PRE posttranscriptional regulation element
M molar PTHs primary Tupaia hepatocytes
mABs monoclonal antibodies
MHBs middle hepatitis B Virus antigen Q
min minute qPCR quantitative PCR
mg milligram
ml millilitre R
mRNA messenger RNA RF resistance factor
MVB multivesicular bodies rcDNA relaxed circular DNA
MxA-protein human myxovirus resistance protein 1 RNA ribonucleic acid
RNP ribonucleoprotein
N rpm revolutions per minute
NES nuclear export signal RT reverse transcription
NLS nuclear localization sequence RT-qPCR reverse transcription quantitative
polymerase chain reaction
NP40 nonident P-40

NPC nuclear pore complex
S
NRE negative regulating element
SD standard deviation
nt nucleotide
SEAP secreted alkaline phosphatase
NTCP sodium taurocholate co-
transporting polypeptide SHBs small hepatitis B virus surface antigen
N-terminus amino-terminus S-HDAg small hepatitis Delta antigen
ssRNA single-stranded RNA
O SV40 simian virus 40
OD optical density
ORF open reading frame T
TAE Tris-acetate EDTA
P TDV tenofovir
PBS phosphate buffered saline TFB transforming buffer
PCH primary chimpanzee hepatocytes TNE Tris-Natrium(sodium)-ETDA
PCR polymerase chain reaction
II
List of abbreviations
V
v/v volume per volume

W
WHO world health organization
WHV woodchuck hepatitis B virus
WMHBV woolley monkey hepatitis B virus
WST water soluble tetrazolium salt
w/w weight per weight
III