Uptake mechanism of Hepatitis B virus into susceptible primary hepatocyte cultures [Elektronische Ressource] = Aufnahmemechanismus des Hepatitis-B-Virus in suszeptible primäre Hepatozyten-Kulturen / vorgelegt von Nicole Kott
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Uptake mechanism of Hepatitis B virus into susceptible primary hepatocyte cultures [Elektronische Ressource] = Aufnahmemechanismus des Hepatitis-B-Virus in suszeptible primäre Hepatozyten-Kulturen / vorgelegt von Nicole Kott

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Angefertigt am Fachbereich 08-Biologie und Chemie in Zusammenarbeit mit dem Institut für Medizinische Virologie der Justus-Liebig-Universität Gießen Uptake mechanism of Hepatitis B Virus into susceptible primary hepatocyte cultures Aufnahmemechanismus des Hepatitis B Virus in suszeptible primäre Hepatozyten Kulturen 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 Fachbereichs 08-Biologie und Chemie der Justus-Liebig-Universität Gießen vorgelegt von Dipl.-Biol. Nicole Kott Gießen 2010 Dekan: Prof. Dr. Volkmar Wolters Gutachter: Prof. Dr. Michael Martin Prof. Dr. Dr. h. c. Wolfram Gerlich Tag der Disputation: 14.01.2011 -Index- Index Abbreviations.......................................................................................... II Summary................................................................................................IV Zusammenfassung .................................................................................. V 1. Introduction ...................................................................................... 1 1.1 Endocytosis.......................................................................................... 1 1.1.1 Entry of pathogens ................................................

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Publié par
Publié le 01 janvier 2010
Nombre de lectures 34
Poids de l'ouvrage 5 Mo

Exrait

Angefertigt am
Fachbereich 08-Biologie und Chemie
in Zusammenarbeit mit dem
Institut für Medizinische Virologie
der Justus-Liebig-Universität Gießen



Uptake mechanism of Hepatitis B Virus into
susceptible primary hepatocyte cultures

Aufnahmemechanismus des Hepatitis B Virus in
suszeptible primäre Hepatozyten Kulturen



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 Fachbereichs 08-Biologie und Chemie
der Justus-Liebig-Universität Gießen



vorgelegt von
Dipl.-Biol. Nicole Kott



Gießen 2010


















Dekan: Prof. Dr. Volkmar Wolters


Gutachter: Prof. Dr. Michael Martin
Prof. Dr. Dr. h. c. Wolfram Gerlich






Tag der Disputation: 14.01.2011
-Index-
Index

Abbreviations.......................................................................................... II

Summary................................................................................................IV

Zusammenfassung .................................................................................. V

1. Introduction ...................................................................................... 1
1.1 Endocytosis.......................................................................................... 1
1.1.1 Entry of pathogens .................................................................................................2
1.2 Pathways of entry into cells.................................................................. 2
1.2.1 Dynamin-dependent endocytosis .........................................................................3
1.2.1.1 Clathrin-mediated endocytosis......................................................................3
1.2.1.2 Caveolin-mediated uptake .............................................................................3
1.2.1.3 Other pathways...............................................................................................4
1.2.2 Dynamin-independent endocytosis ......................................................................4
1.2.2.1 Phagocytosis....................................................................................................4
1.2.2.2 Macropinocytosis............................................................................................5
1.2.2.3 CLIC-GEEC .....................................................................................................6
1.2.2.4 Other pathways...............................................................................................6
1.2.3 pH dependence of viral infection ..........................................................................7
1.2.4 Role of the cytoskeleton for endocytic pathways .................................................7
1.3 Hepatitis B virus (HBV) .......................................................................8
1.3.1 Taxonomy ...............................................................................................................8
1.3.2 Transmission and disease......................................................................................8
1.3.3 Prevention and therapy .........................................................................................9
1.3.4 Morphology...........................................................................................................10
1.3.5 Genome.................................................................................................................12
1.3.6 Viral life-cycle....................................................................................................... 14
1.3.7 Model systems for HBV infection ....................................................................... 16
1.4 Aim of this work..................................................................................17

2. Material ........................................................................................... 18
2.1 Buffer, solutions and media ............................................................... 18
2.1.1 Buffers...................................................................................................................18
2.1.2 Solutions ...............................................................................................................18
2.1.3 Media.....................................................................................................................19
2.2 Chemicals ..........................................................................................20
2.3 Inhibitors............................................................................................21
2.3.1 Pharmacological inhibitors..................................................................................21 -Index-
2.3.2 myr preS1-HBV peptide.......................................................................................21
2.4 Viruses................................................................................................21
2.4.1 HBV.......................................................................................................................21
2.4.2 HBsAg ...................................................................................................................21
2.4.3 Control viruses ..................................................................................................... 21
2.5 Cell Culture........................................................................................ 22
2.5.1 Cell culture systems .............................................................................................22
2.5.2 Cells.......................................................................................................................22
2.5.2.1 Primary Tupaia hepatocytes (PTH) ............................................................22
2.5.2.2 HepG2 ...........................................................................................................23
2.5.2.3 HepaRG.........................................................................................................23
2.5.2.4 BHK ...............................................................................................................23
2.5.2.5 CV-1 ...............................................................................................................23
2.5.2.6 Huh-7.............................................................................................................23
2.5.2.7 HeLa ..............................................................................................................23
2.6 Antibodies and –sera ......................................................................... 23
2.6.1 Primary antibodies and –sera .............................................................................23
2.6.2 Secondary antibodies...........................................................................................24
2.7 Fluorescent dyes ................................................................................ 24
2.8 DNA-and protein standards ............................................................... 24
2.9 Plasmids ............................................................................................ 24
2.10 Commercial kits................................................................................. 24
2.11 PCR .................................................................................................... 24
2.11.1 X-PCR....................................................................................................................24
2.11.2 cccDNA-PCR.........................................................................................................24
2.11.3 SV40-PCR.............................................................................................................25
2.11.4 Primer and Hybridisation probes (Hybprobes).................................................25
2.11.5 Mastermixes .........................................................................................................25

3. Methods ...........................................................................................26
3.1 Cell culture......................................................................................... 26
3.1.1 Isolation of PTH ...................................................................................................26
3.1.2 Cultivation of stable cell lines..............................................................................27
3.1.2.1 BHK21, CV-1, HeLa, HepG2, Huh-7 ...........................................................27
3.1.2.2 HepaRG.........................................................................................................27
3.2 Virus preparation .............................................................................. 27
3.2.1 HBV.......................................................................................................................27
3.2.2 SFV ........................................................................................................................27
3.2.3 SV40..................................................................................................................... 28
3.2.4 SeV........................................................................................................................ 28
3.3 Hemagglutination (HA)-assay............................................................ 28 -Index-
3.4 Plaque assay....................................................................................... 28
3.4.1 SFV ....................................................................................................................... 28
3.5 Horseradish peroxidase (HRP)-Assay................................................ 28
3.6 Competent bacteria............................................................................ 29
3.7 Transformation.................................................................................. 29
3.8 Plasmid isolation ............................................................................... 29
3.9 Transfektion ...................................................................................... 29
3.9.1 FuGene..................................................................................................................29
3.9.2 Amaxa ...................................................................................................................29
3.9.3 Baculovirus-mediated transduction .................................................................. 30
3.10 Binding and uptake studies................................................................30
3.10.1 HBsAg .................................................................................................................. 30
3.10.2 Phagocytosis ........................................................................................................ 30
3.11 Infection assays..................................................................................30
3.11.1 HBV...................................................................................................................... 30
3.11.1.1 cccDNA isolation and Hirt extraction ......................................................... 31
3.11.1.2 Urea-Test.......................................................................................................32
3.11.2 SFV ........................................................................................................................32
3.11.3 SV40......................................................................................................................32
3.11.4 SeV.........................................................................................................................32
3.12 ELISA................................................................................................. 33
3.13 Immunostaining ................................................................................ 33
3.14 PCR .................................................................................................... 33
3.14.1 X-PCR....................................................................................................................33
3.14.2 cccDNA-PCR.........................................................................................................34
3.14.3 SV40-PCR.............................................................................................................34
3.14.4 Agarose gel electrophoresis of PCR products.....................................................34

4. Results .............................................................................................35
4.1 HBsAg binding and uptake in PTH ..................................................... 35
4.2 Endocytosis vs. plasma membrane fusion of HBV and control viruses36
4.2.1 Semliki Forest Virus (SFV)................................................................. 36
4.2.2 Simian Virus 40 (SV40) ..................................................................... 37
4.2.3 Sendai Virus (SeV) ............................................................................. 38
4.2.4 Inhibitors used to block certain cellular endocytic steps.................... 39
4.3 Dynamin-dependent endocytosis ....................................................... 45
4.3.1 Clathrin-mediated endocytosis ...........................................................................45
4.3.2 Caveolin-mediated uptake.................................................................................. 48
4.4 Dynamin-independent endocytosis.................................................... 52
4.4.1 Phagocytosis .........................................................................................................52
4.4.2 Macropinocytosis .................................................................................................53 -Index-
4.5 pH-dependence of viral infection ....................................................... 56
4.6 Cytoskeleton ...................................................................................... 59
4.7 Retrograde pathway........................................................................... 67
4.8 Transfection of PTH ........................................................................... 70
4.8.1 Lipofection vs. electroporation............................................................................70
4.8.2 Transduction by Baculoviruses ........................................................................... 71

5. Discussion........................................................................................ 72
5.1 Binding and uptake of HBsAg............................................................. 72
5.2 Difficulties in studying endocytic processes ....................................... 72
5.2.1 Methods for the classification of endocytic mechanisms..................................73
5.3 Endocytosis vs. plasma membrane fusion of HBV.............................. 75
5.4 Dynamin-dependent endocytosis ....................................................... 76
5.4.1 Clathrin-mediated endocytosis ...........................................................................76
5.4.2 Caveolin-mediated endocytosis........................................................................... 77
5.5 Dynamin-independent endocytosis.................................................... 78
5.5.1 Phagocytosis .........................................................................................................78
5.5.2 Macropinocytosis .................................................................................................79
5.6 pH-dependence of HBV infection....................................................... 81
5.7 Role of the cytoskeleton for HBV infection......................................... 82
5.8 Retrograde pathway involved in viral uptake ..................................... 83
5.9 Future issues......................................................................................84

6. References .......................................................................................85

7. Publications ..................................................................................... 97

8. Acknowledgements..........................................................................98

9. Declaration ......................................................................................99
-Abbreviations- I
Abbreviations

A
ad fill up to DHBV Duck Hepatitis B Virus
Ad2/3/5 Adenovirus type 2/3/5 DMEM Dulbecco´s Modified Eagle
AP adaptor protein Medium
Arf1/6 ADP-ribosylation factor 1/6 DMSO Dimethyl sulfoxide
ASHV Arctic Squirrel Hepatitis dn dominant negative
Virus DNA Desoxyribonucleotide acid
DR1/2 direct repeat 1/2
B
BafA1 Bafilomycin A1 E
bp basepair ε encapsidation signal
BFA Brefeldin A E. coli Escherichia coli
BSA bovine serum albumin EE early endosome
EIPA (5-[N-ethyl-N-isopropyl]
C amiloride)
cav-1/2/3 caveolin-1/2/3 EM electron microscopy
cccDNA covalently closed circular Enh enhancer
DNA Eps15 epidermal growth factor
CCP clathrin-coated pit receptor substrate 15
CCV clathrin-coated vesicle ER endoplasmic reticulum
Cdc42 cell division control protein ERAD ER-associated protein
42 homolog degradation-machinery
CD59 complement regulatory
protein F
CHBV Crane Hepatitis B Virus FCS fetal calf serum
ChHBV Chimpanzee Hepatitis B Virus Fig figure
CI clathrin-independent
CLIC clathrin-independent G
carriers g multiples of gravitational
CME clathrin-mediated endocytosis acceleration on earth
C-terminus carboxy terminus GE genome equivalents
CtxB Cholera Toxin B GEEC GPI-AP enriched
CytoD Cytochalasin D endosomal compartment
GiHBV Gibbon Hepatitis B Virus
D GoHBV Gorilla Hepatitis B Virus
d distilled GPI-AP glycosyl phosphatidyl-
Da Dalton -inositol-anchored protein
-Abbreviations- II
GRAF-1 GTPase regulator L
associated with focal l liter
adhesion kinase-1 L lysosome
GRE glucocorticoid-responsive LB medium Luria Broth medium
element LHBs Large Hepatitis B surface
GSHV Ground Squirrel Hepatitis protein
Virus
M
H µ micro
h hour m milli/meter
HA hemagglutination assay M molar
HBcAg Hepatitis B core antigen MßCD methyl-beta-cyclodextrin
HBeAg Hepatitis B e antigen MHBs Middle Hepatitis B surface
HBsAg Hepatitis B surface antigen protein
HBxAg Hepatitis B x antigen MHC-1 major histocompatibility
HBV Hepatitis B Virus complex 1
HCC Hepatocellular carcinoma min minutes
HGM hepatocyte growth medium MLV Murine Leukemia Virus
HHBV Heron Hepatitis B Virus MOI multiplicity of infection
HIV-1 Human Immunodefiency mRNA messenger ribonucleic acid
Virus 1 MT microtubule
HN hemagglutitin-neuraminidase MTOC microtubule organizing
HPV-16 Human Papilloma Virus 16 center
HRP horseradish peroxidase MVBs multivesicular bodies
HSPGs heparan sulfate MW molecular weight
proteoglycans
HSV-1 Herpes Simplex Virus 1 N
n nano
I NLS nuclear localization signal
ID internal identification Noco Nocodazole
number NPC nuclear pore complex
IL-2R-β β-chain of the interleukin-2 NRE negative regulating element
receptor nt nucleotide
IFN interferon N-terminus amino terminus

J O
Jas Jasplakinolide OuHBV Orang-Utan Hepatitis B Virus
ORF open reading frame
K
k kilo
-Abbreviations- III
P
PBS phosphate buffered saline T
PCR polymerase chain reaction TEMs tetraspanin-enriched
PDH primary duck hepatocytes microdomains
PEG polyethylene glycol TGN trans Golgi network
PFA paraformaldehyde TNE Tris-Natrium(sodium)-EDTA
PFU plaque forming units
pg pregenomic V
PHH primary human hepatocytes VacA Helicobacter pylori
p.i. post infection vacuolating toxin A
PM plasma membrane v-ATPases vacuolar-type ATPases
pol polymerase VSV Vesicular Stomatitis Virus
pr primer domain
PRE posttranscriptional regulatory W
element
WHO World Health Organization
preS1/2 preSurface 1/2
WHV Woodchuck Hepatitis B
PTH primary Tupaia hepatocytes
Virus

WMHBV Wolly Monkey Hepatitis B
R Virus
Rac1 Ras-related C3 botulinum
toxin substrate 1 Other
rcDNA relaxed circular DNA
# Number
RE recycling endosome
RFP red fluorescent protein
RhoA Ras homolog gene family
member A
RNA ribonucleic acid
rpm rounds per minute
RT reverse transcription

S
sec second
S surface
SARS severe acute respiratory
syndrome
SeV Sendai Virus
SFV Semliki Forest Virus
SHBs Small Hepatitis B surface
protein
SV40 Simian Virus 40
-Summary- IV
Summary
Hepatitis B virus (HBV) can infect only a few cell types including primary Tupaia
hepatocytes (PTH) which were used in this work. The aim of the study was to characterize
the early steps of HBV infection using pharmacological inhibitors and confocal microscopy.
The distinction between endocytosis in general and direct fusion with the plasma
membrane was made by using hypertonic sucrose concentrations. The fusion of Sendai
virus (SeV) with the plasma membrane and its infectivity was not affected, whereas the
infection of control viruses known to be taken up via endocytosis, e.g. Semliki Forest virus
(SFV) and Simian virus 40 (SV40), was impeded. The HBV infection was inhibited in a
dose-dependent manner, suggesting an endocytic uptake. The dynamin inhibitor Dynasore
completely inhibited the entry of SFV (clathrin-mediated endocytosis) and SV40 (caveolin-
mediated endocytosis), but had no effect on HBV suggesting a dynamin-independent
internalization. Furthermore, the inhibitor of macropinocytosis EIPA did not impair HBV
infection. HBV was also not able to stimulate the macropinocytic activity of PTH. Also
phagocytosis could be ruled out as an entry mechanism. The actin inhibitors Jasplakinolide
(actin-stabilisation) or Cytochalasin D (actin-depolymerisation) resulted in a partial
inhibition of the early steps of HBV infection. The intracellular transport of HBV could be
constrained by utilization of Nocodazole, an inhibitor of microtubule-polymerisation. HBV
does not require an acidic pH within the endosomal compartments for the initiation of a
productive infection cycle and therefore differs from many other viruses that are
internalized via endocytosis (e.g. SFV, which was tested as control virus) and also from the
related hepatitis B virus of the duck. No parallels to the retrograde pathway of SV40 were
observed, since the HBV infection showed no sensitivity towards inhibitors of specific
intracellular transport mechanisms (Brefeldin A), ER protein folding and quality factors
(MG-132) and the translocation process to the cytosol (Thapsigargin). Together with
previous data it can be concluded that HBV is internalized via a not yet known, possibly
virus-induced, endocytic uptake mechanism, which is clathrin-, caveolin-, lipid raft- and
dynamin-independent and differs from the process of phagocytosis and macropinocytosis.