Hepatitis C virus entry and cell-cell transmission: implication for viral life cycle and antiviral treatment

170 Pages·2017·7.2 MB·French

by  Fei Xiao

UNIVERSITÉ DE STRASBOURG

ÉCOLE DOCTORALE 414, ED des Sciences de la Vie et de la Santé
LABORATOIRE: INSERM U1110

THÈSE

présentée par:
Fei XIAO

soutenue le : 28 Juillet 2014

pour obtenir le grade de: Docteur de l’Université de Strasbourg
Discipline/ Spécialité: Virologie: aspects moléculaires et médicaux

Hepatitis C virus entry and cell-cell
transmission: implication for viral life
cycle and antiviral treatment

THÈSE dirigée par :

Mr BAUMERT Thomas Professeur, Université de Strasbourg

RAPPORTEURS :

Mr NASSAL Michael Professeur, University Hospital Freiburg

Mr FÉRAY Cyrille Professeur, Inserm U955, Hôpital Henri Mondor

AUTRE MEMBRE DE JURY (EXAMINATEUR) :

Mr RICCI Romeo Professeur, IGBMC, Université de Strasbourg
Where there’s a will, there’s a way
.

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Acknowledgement
First of all, I would like to thank my supervisor, Prof. Thomas Baumert, for his
guidance and support since my first day in the lab. I’ve learned a lot from his
enthusiasm for science, spirit of innovation and hard-working attitude and will follow
his footsteps to develop my career.
Many thanks to my thesis committee members, Prof. Michael Nassal, Prof. Cyrille
Férray and Prof. Romeo Ricci, who squeeze their precious time to participate in my
thesis defense.
I would like to express my heartful thanks to Mirjam Zeisel, who gave me a lot of
detailed guidance on designing the experiments and writing the manuscripts and
encouraged me to cheer up when I felt depressed and hopeless. Like my older sister,
she shared my joy and sorrow and gave me strength to move forward.
I express my gratitude to Christine Thumann, who helped me to adapt to the new
environment when I came to the lab and taught me a lot of experimental skills. She
not only made a lot of contributions to my projects but also provided many practical
tips and information for my living in Strasbourg.
I express my sincere thanks to senior staffs, Catherine S., Joachim, Heidi, Laurent,
Eric R., Samira, Christiane and Françoise for their support!
I acknowledge Eric S., Laura, Sarah, Charlotte, Jochen and Nicolas B. for doing
experiments and providing technical support for my projects.
I give my special thanks to Catherine C., Anne, Sigis, Jérémy and Patricia for their
help in administrative and technical issues.
I feel grateful to my colleges Rajeev, Dan, Olga, Nicolaas, Eloi, Catherine F.,
Simonetta, Sophie P., Lamine, Emilie, Antonina, Thomas D., Sylvie and Géraldine for
the friendship and support. I will never forget my previous colleagues Isabel, Patric,
Sophie K., Laetitia, Daniel, Marine, Nauman, Céline and Roxane, although they have
left the lab.
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My gratitude to my Chinese colleagues Bin and Tao as well as the previous ones
Yifan, Ke and Hao, who gave me full support and relieved my homesickness.
Words are not enough to express my thanks to my parents, who gave me life,
brought me up and supported me without any conditions.
Finally, I would like to thank Inserm, the University of Strasbourg, the French
government and the European Union for providing me scholarships and salary to
support my research.
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Table of Content
List of figures ............................................................................................................. 6
Abbreviations ............................................................................................................. 7
Résumé....................................................................................................................... 9
Introduction ............................................................................................................. 15
History of HCV ........................................................................................................ 17
Epidemiology and transmission of hepatitis C ........................................................ 17
Diagnosis of hepatitis C .......................................................................................... 18
Pathogenesis of hepatitis C .................................................................................... 19
Viral genome and genetic diversity ......................................................................... 20
HCV cell culture models ......................................................................................... 23
HCV animal models ................................................................................................ 25
HCV entry and cell-cell transmission ...................................................................... 27
HCV translation, replication and assembly ............................................................. 31
Innate and adaptive immune responses to HCV .................................................... 33
Treatment of chronic hepatitis C ............................................................................. 34
Aims of the study ................................................................................................... 37
Results..................................................................................................................... 38
Part 1: Synergy of enty inhibitors with direct-acting antivirals uncovers
novel combinations for prevention and treatment of hepatitis C ....................... 42
Introduction ............................................................................................................. 43
Publication nº1: Synergy of enty inhibitors with direct-acting antivirals uncovers
novel combinations for prevention and treatment of hepatitis C ............................. 44
Abstract ............................................................................................................... 45
Significance of the study ..................................................................................... 46
Introduction ......................................................................................................... 47
Material and methods ......................................................................................... 48
Results ................................................................................................................ 51
Discussion .......................................................................................................... 58
References ......................................................................................................... 63
Figure legends .................................................................................................... 68
Figures ................................................................................................................ 71
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Supplementary data ............................................................................................ 77
Part 2: Hepatitis C virus cell-cell transmission and resistance to
direct-acting antiviral agents ................................................................................. 89
Introduction ............................................................................................................. 90
Publication nº2: Hepatitis C virus cell-cell transmission and resistance to
direct-acting antiviral agents ................................................................................. 91
Abstract ............................................................................................................... 93
Author summary .................................................................................................. 94
Introduction ......................................................................................................... 95
Material and methods ......................................................................................... 97
Results .............................................................................................................. 101
Discussion ........................................................................................................ 106
References ....................................................................................................... 109
Figure legends and tables ................................................................................. 115
Figures and supplementary tables .................................................................... 124
Conclusions and perspectives ............................................................................ 136
References ............................................................................................................ 140

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List of Figures
Résumé Figure : Combinaisons d’inhibiteurs d'entrée et des antiviraux directs dans
l’infection par le HCV ................................................................................................ 14
Figure 1: Global prevalence of HCV ........................................................................ 18
Figure 2: Neutralizing antibody and HCV RNA in resolving and chronic HCV
infection .................................................................................................................... 19
Figure 3: Outcomes associated with HCV infection ................................................. 20
Figure 4: Phylogenetic tree of members of the family Flaviviridae ........................... 21
Figure 5: HCV genome ............................................................................................ 22
Figure 6: Evolutionary tree of HCV genotypes 1-7 .................................................. 22
Figure 7: HCV cell culture model ............................................................................. 24
Figure 8: Human liver uPA-SCID mouse model with HCV infection ........................ 26
Figure 9: HCV entry ................................................................................................. 27
Figure 10: HCV host factors .................................................................................... 29
Figure 11: Cell-free transmission and cell-cell transmission .................................... 31
Figure 12: Different mechanisms of virus cell-cell transmission .............................. 31
Figure 13: HCV life cycle ......................................................................................... 32

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Abbreviations

BHV Bat hepacivirus
CARD Caspase recruitment domain
CC Cytotoxic concentration
CD81 Cluster of differentiation 81
CI Combination index
CLDN Claudin
CTRL Control
DAA Direct-acting antiviral
EGFR Epidermal growth factor receptor
EIA Enzyme immunoassay
EMCV Encephalomyocarditis virus
EphA2 Erythropoietin-producing hepatocellular receptor tyrosine kinase
class A2
HA Human albumin
HAV Hepatitis A virus
HBV Hepatitis B virus
HCC Hepatocellular carcinoma
HCV Hepatitis C virus
HCVcc Cell culture-derived HCV
HCVpp Hepatitis C virus pseudoparticle
HIV Human immunodeficiency virus
HTA Host-targeting agent
HTEI Host-targeting entry inhibitors
IC Inhibitory concentration
IFN Interferon
IL Interleukin
IRES Internal ribosome entry site
ISG Interferon-stimulated gene
LOQ Limit of quantification
LT Liver transplantation
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mAb Monoclonal antibody
MAV Mitochondrial antiviral signaling protein
miRNA microRNA
MLV Murine leukemia virus
MOA Mechanism of action
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NANB Non-A, non-B
NPC1L1 Niemann Pick C1-Like 1
NPHV Non-primate hepacivirus
NTR Non translated region
OCLN Occludin
ORF Open reading frame
PEG-IFN Pegylated interferon
PCR Polymerase chain reaction
PHH Primary human hepatocyte
PKI Protein kinase inhibitor
PKR Protein kinase R
RBV Ribavirin
RHV Rodent hepacivirus
RIG-I Retinoic acid inducing interferon gene I
RLR RIG-I like receptor
SOC Standard of care
SR-BI Scavenger receptor class B type I
SVR Sustained viral response
TLR Toll like receptor
UTR Untranslated region
WHO World health organization
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Résumé

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