Table Of ContentPhD program in Translational and Molecular Medicine
DIMET
Studies on the mechanism of action of
antiviral agents targeting the replication
complex of hepatitis C virus
Coordinator: Prof. Andrea Biondi
Tutor: Dr. Raffaele De Francesco
Dr. Veronica Reghellin
Matr. No. 078959
XXVI CYCLE
ACADEMIC YEAR 2012-2013
“Il processo di una scoperta scientifica
è un continuo conflitto di meraviglie”
(A. Einstein)
To Andrea and my family
Table of Contents
CHAPTER I GENERAL INTRODUCTION…………………………………. ………..….1
A. Hepatitis C Virus……………………………………………..………..………….. 2
1. The discovery of the Hepatitis C Virus…………………………………….…. 2
1.1. Classification of Hepatitis C Virus……………………….……………….. .3
2. Molecular organization of Hepatitis C Virus……………………….……... 4
Genomic heterogeneity and classification systems of HCV…… ...... 4
2.1. Genome Organization……………………………………………………....... 6
2.2.1. Untranslated RNA Segments…………………………………. ……7
2.2.2. Structural proteins …………………….………………………… ….10
2.2.3. Non structural proteins ….……………………………………… ..13
2.2.3.1.NS5A: structure, phosphorylation and interaction . …19
3. The viral lifecycle ……………………………………………………………………. 28
3.1. The Viral Particle…………………………………………………………..…... 29
3.2. Entry and Uncoating…………………………………………………………… 30
3.3. Translation and Polyprotein Processing……………….…………….. 33
3.4. HCV RNA Replication…………………….…………………………………….34
3.4.1. The HCV RNA Replication ............................................ 36
3.4.2. The HCV-induced membranous web………………….. ...... 37
3.4.3. Known cellular factors of HCV replication: the important
role of PI-4 Kinase type IIIα……………………… ............... …41
3.5. HCV Assembly and Release………………………………………………… 47
4. Lipid metabolism and HCV Replication……………………………………..49
5. Model to study HCV ……………………………………………………………….. 55
5.1. Animal models………………………………………………………………….. 55
5.2. The replicon system…………………………………………………………… 56
5.3. HCV pseudoparticles (HCVpp) …………………………………………... 58
5.4. Cell-culture derived HCV (HCVcc) ……………………………………… 59
B. Hepatitis C Virus infection: clinical aspects………………………… 61
1. Epidemiology, Geographical Distribution and Transmission… . 61
2. Natural history of Infection…………………………………………………… 66
2.1. Acute Hepatitis C……………………………………………………………….. 67
2.2. Chronic Hepatitis C……………………………………………………………. 68
2.3. Cirrhosis and Hepatocellular Carcinoma………………………….… 68
3. Current management of Hepatitis C: diagnostic tools and
treatment responses………………………………………………………….… 70
4. Treatment of HCV infection………………………………..……………….. 74
4.1. The era of Direct-Acting Antivirals…………………………………….. 75
4.2. Daclatasvir: the first in class of NS5A Replication Complex
inhibitors…………………………………………………………………………… 83
4.3. Host targeting agents (HTA) and other antiviral
treatments ………………………………………………………………………… 88
5. Still unmet clinical needs in Hepatitis C: diagnostic tools,
biomarkers of disease progression and new
therapies……………………………………………………………………………. . 93
C. Scope of the thesis…………………………………………………………….. 97
D. Chapter I-References……………………………………………………….. 101
CHAPTER II. METABOLISM OF PHOSPHATIDYLILINOSITOL 4-KINASE
IIIA-DEPENDENT PI4P IS SUBVERTED BY HCV AND IS TARGETED BY A
4-AMINO QUINAZOLINE WITH ANTIVIRAL ACTIVITY...................... 125
A. Abstract…………………………………………………………….……………… .. 126
B. Introduction…………………………………………………………………….. ... 127
C. Results………………………………………………………………………….….. .. 131
1. Compound AL-9 inhibits HCV replication in vitro…………..…. . 131
2. AL-9 is an inhibitor of PI4KIIIα………………………………………..... . 133
3. HCV alters the intracellular and plasma membrane
distribution of PI4P…………………………………………………………… 142
4. Al-9 inhibits PI4KIIIα in HCV replicating cells………………….…. 147
5. PI4KIIIα inhibition by Al-9 alters sub-cellular distribution of
N55A………………………………………………………………………………… 150
D. Discussion………………..…………………………………………………….… ... 153
E. Materials and Methods………… .... ………………………………………….160
1. Reagents and plasmids………………………………………………… …..160
2. Cell lines and culture conditions…………………………………… …..161
3. Replication and infection assays……………………………………… ..161
4. Expression and purificationof the catalytic domain of
PI4KIIIα…………………………………………………………………………….. 163
5. In vitro kinase assay………………………………………………………….. 164
6. Indirect immunofluorescence…………………………………………… 164
7. siRNA silencing……………………………………………………………….... 166
8. T7-driven HCVpolyprotein expression………………………………. 167
F. Supporting Information………… ... ………………………………………….168
G. Acknowledgments………………………………… ... …………..…………….177
H. Chapter II – References…………………………… ... ……………………….179
CHAPTER III. NS5A INHIBITORS INTERFERE WITH NS5A-PI4KIIIα
COMPLEX FORMATION AND CAUSE A CONCOMITANT DECREASE OF
PI4P AND CHOLESTEROL IN THE HCV MEMBRANOUS WEB
……………………………………………………………..……………………………………… 185
A. Abstract…………………………………………………………….……………… ... 186
B. Introduction……………………………………………………………………… ... 187
C. Results……………………………………………………………………………… .... 191
1. Chemically diverse NS5A inhibitors block NS5A
hyperphosphorylation ……………………………………………………… 191
2. NS5A inhibitors promote formation of “large cluster” NS5A
phenotype………………………………………………………………………… 198
3. NS5A inhibitors interfere with the enrichment of PI4P in HCV
replication membranes…………………………………………………….. 201
4. NS5A inhibitors interfere with the NS5A-PI4KIIIα protein
complex formation………………………………………………………… …205
5. Both PI4KIIIα- and NS5A inhibitors reduce cholesterol
concentration in the HCV-induced membranous web………. 211
D. Discussion………………..……………………………………………………. ... …218
E. Materials and Methods…………………………… ... ……………………….224
1. Reagents and Plasmids……………………………… ………………………224
2. Cell lines and culture conditions…………………… ……………….….225
3. Replication assays…………………………………………………………….. 225
4. Protein expression with the Vaccinia Virus T7
infection/transfection system, immunoprecipitation and
Western Blotting………………………………………………………………. 226
5. Indirect immunofluorescence………………………………...……… …228
F. Chapter III – References………………………………………………..… ….230
CHAPTER IV. SUMMARY, CONCLUSIONS AND TRANSLATIONAL
IMPACT OF THE PROJECTS …………………………………………………………. .241
A. Discovery of HCV inhibitors targeting host factors…………. …242
B. Elucidation of mode of action of NS5A replication complex
inhibitors………………………………………………………………………… …..244
C. Chapter IV – References…………………………………………..…… ... ….249
PUBLICATIONS ………………………………………………………. …………………….253
CHAPTER I
GENERAL INTRODUCTION
1
