Characterization of the infectivity determinants of the envelope proteins of hepatitis B-virus [Elektronische Ressource] / presented by Yi Ni
170
pages
English
Documents
2010
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170
pages
English
Ebook
2010
Obtenez un accès à la bibliothèque pour le consulter en ligne En savoir plus
Publié par
Publié le
01 janvier 2010
Nombre de lectures
18
Langue
English
Poids de l'ouvrage
26 Mo
Publié par
Publié le
01 janvier 2010
Nombre de lectures
18
Langue
English
Poids de l'ouvrage
26 Mo
Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
presented by
Yi Ni
born in: Fujian, P.R. China
Oral-examination:
Characterization of the infectivity determinants of the envelope
proteins of Hepatitis B Virus
Referees: Prof. Dr. Stephan Urban r. Ursula Klingmüller Index I
Index
Summary………………………………………………………………………….…….……..1
Zusammenfassung……………………………………………………………………...…….3
1. Introduction…………………………………………………..……………..……….……..5
1.1 Hepatitis B virus biology..………………………………………………..….…….....5
1.1.1 Classification of viruses within the Hepadnavirus family…………………..………5
1.1.2 Serotypes and genotypes………………………………………………….……….5
1.1.3 Virus structure…………………………………….………………………………7
1.1.4 Genome structure and organization………………………………………...……..7
1.1.5 Primary attachment and receptor mediated binding …………………………..…..9
1.1.6 Fusion with cellular membrane…………………………………….………….…12
1.1.7 Nucleocapsid transportation……………………………………………..………12
1.1.8 cccDNA formation…………………………………………………………........13
1.1.9 Transcription………………………………………………………………….…13
1.1.10 Translation of viral proteins and co/post-translational modification…………....14
1.1.11 Genome replication……………………………….……………………….…....15
1.1.12 Assembly and virus release…………………………………………………...…15
1.2 The HBV entry inhibitor: Myrcludex B…………………………………….………17
1.3 Host and organ tropism of hepadnaviruses….…………………………..…………19
1.4 Cellular factors needed for infection………………………………………..………19
1.5 The HBV envelope and known viral infectivity determinants…………………….20
1.6 Animal and cellular models for HBV infection……………...……………..………24
1.7 Aim of this work ………………………………………………………………..……27
2. Results………………………………………………………………………………..……29
2.1 Optimization of virus production and infection assay………………………….…29 Index II
2.1.1 Optimization the viral production by transfection of complementary expression
plasmids………………………………………………………..……..……….....29
2.1.2 Kinetics of virus production following co-transfection….……….……...….….…30
2.1.3 Anti-core IF staining of infected HepaRG cells……..………………………...….31
2.1.4 Quantification of the infection by secreted viral marker and the number of infected
cells……………………………………………………………………..……......33
2.1.5 Optimization of the infection-the effect of DMSO………………..……….….…34
2.1.6 Optimization of the infection-the effect of “spin inoculation”……………...……37
2.1.7 Optimization of the infection-the effect of PEG……………………..……….…38
2.1.8 Optimization of the infection-the effect of EGTA…………………………...….40
2.1.9 The effect of passage number on HBV infection ………………………………..41
2.2 The N-terminus of preS1 domain serves a critical function for virus
infectivity…………………………………………………………………………….46
2.2.1 Infectivity of genotypically L-protein pseudotyped HBV particles…………….…48
2.2.2 Infectivity of HBV carrying chimeric L proteins from genotypes D and B………50
2.2.3 Infectivity of HBV carrying chimeric L protein with His-6 tag……..……….……52
2.2.4 Infectivity of HBV with different myristoylation motifs ...…………………….…54
2.2.5 Infectivity of HBV carrying chimeric L protein of different genotype………....…56
2.2.6 Characterization of inhibitory peptides bearing mutations in the N-terminus …....58
2.2.7 The role of myristoylation in HBV entry………………………………………....64
2.2.8 A possible role of preS aa 49-78 in virus entry..…………….……………..……...67
2.3 Using M protein-free virus to dissect the role of preS2 for assembly and infectivity
of Hepatitis B virus ……………………………………………………………....…70
2.3.1 M-free system with independent expression of S and L-protein without loss of
infectivity.…………………………………………………………………….....70
2.3.2 No specific sequence in preS2/114-163 is needed for virus infectivity but a length
dependent linker within this region is important for viral assembly …………..…73 Index III
2.3.3 M-protein-deficient HBV with a randomized preS2-sequence in its L-protein
properly assembles and is infectious in HepaRG cells and PHH……………...…75
2.4 Analysis of the role of the S domain in the L protein and the S protein for
assembly and infectivity of HBV………………………………………………..….79
2.4.1 The effect of cysteine mutations in the S and the L protein on protein expression
and virus secretion………………………………….…..…………….……….....80
2.4.2 The effect of cysteine mutations in the S and the L protein on virus assembly.…..81
2.4.3 The effect of cysteine mutations in the S and the L protein on HBV infectivity.....83
2.4.4 The effect of cysteine mutations on the topology of the L-protein……...….….…85
2.4.5 The heparin-binding activity of the HBV particles with cysteine mutations…..…..88
3. Discussion…………………………………….…………………………….…….………92
3.1 Analysis of different parameters on their effect on HBV infection in HepaRG cells .….92
3.2 The role of N-terminus of L-protein in HBV assembly………………….………….…95
3.3 The role of preS2 domain in HBV assembly and infectivity………….………...……...100
3.4 The role of S-domain in HBV entry………………………………………………..…102
3.5 Model for HBV attachment and receptor binding …………………………...…….…104
4. Materials and Methods………………………………………………………….………107
4.1 Materials ……………………………………………………………..………..……107
4.1.1 Instruments, consumables, software, and reagents…………………………...…107
4.1.2 Oligonucleotides………………………………………………………...........…111
4.1.3 Plasmids……………………………………………………………………...…117
4.1.4 Peptides…………………………………………………………………...……123
4.1.5 Cells…………………………………………………………………………….124
4.2 Methods………………………………………………………………….……….…125
4.2.1 General Cloning strategy……………………………………………………..…125
4.2.2 Agarose electrophoresis……………………………………………………...…125
4.2.3 Gel exaction of DNA fragments……………………...…………….………...…125 Index IV
4.2.4 Polymerase Chain Reaction (PCR) ……………………………………………..126
4.2.5 Competent cells preparation and transformations………………………………126
4.2.6 DNA sequencing…………………………………………………………….…127
4.2.7 Plasmid preparations...……………………………………………………….…127
4.2.8 SDS-PAGE……………………………………………………….……………127
4.2.9 Western blot……………………………………………………………………128
4.2.10 Immunoprecipitation……………………………………………………….…128
4.2.11 Cultivation of HuH-7 cells…………………………………………………….129
4.2.12 Cultivation and infection of primary human hepatocytes and HepaRG cells
………………………………………………………….…………………...…129
4.2.13 Transfection of HuH-7 using polyethylenimine………………..………..……..131
4.2.14 Production of HBV particles…………………..………………………………131
4.2.15 Purification and concentration of virus by PEG-precipitation……………...….131
4.2.16 CsCl-gradient centrifugation and DNA dot-blot analysis…...………..……..….132
4.2.17 Immunofluorescence analyses of HBcAg expression in infected cells....……….132
4.2.18 HBsAg and HBeAg measurement in the supernatants of infected cells……….132
4.2.19 Heparin binding assay ……………………………………………………...…132
4.2.20 lentivirus production and transduction of HepaRG cells………………………133
References list………………………………………………………………….………..….134
Abbreviations …………………………………………………………………….……..….153
Supplemental……………………………………………………………………….……....156
Assurance of research…………………………………………………….……………...…161
Curriculum Vitae …………………………………………………………………….…….162
Publications………………………………………………………………………...………163
Acknowledgement……………………………………………………………………….…164 Summary 1
Summary
The human Hepatitis B virus (HBV) is a small hepatotropic DNA virus, which consists of a
nucleocapsid and an envelope with three membrane-embedded surface proteins (large (L),
middle (M), and small (S)). While the S protein is required for budding and is the major
component of the envelope, the L protein is crucial for infectivity. Several infectivity
determinants have previously been described: (i) The N-terminal myristic acid moiety together
with aa 2-48 are indispensable for virus infectivity and specifically bind a yet unknown receptor.
myrConsistent with this, a peptide HBVpreS/2-48 , composed of a myristoyl group and the N-
terminal 47 residues of the L protein, inhibits HBV entry at picomolar concentrations. (ii) Amino
acids 49-78 and the first transmembrane domain of the L protein are also required for virus
infectivity. However, their function is not clear. (iii) Recently, it has been shown that the S-
domain, which is common to all three surface proteins, also contains an infectivity determinant
in its antigenic loop. Currently, it is not fully understood how the virion orchestrates these
infectivity determinants during entry process and how exactly the preS-derived peptide
myrHBVpreS/2-48 interferes with virus entry. The major obstacle, restricting such investigations
for a long time, was the lack of easily accessible in vitro infection systems. A recently established
human hepatoma cell line (HepaRG), susceptible for HBV infection upon differentiation in vitro,
resolved this issue and allowed us to analyze the role of the HBV envelope proteins in virus
entry.
In the present work, several approaches were undertaken: (i) systematic optimization of He
