Sultan ALI

208 Pages·2013·18.34 MB·English

by  Sultan Ali

UNIVERSITÉ DE STRASBOURG

École Doctorale des Sciences de la vie et de la Santé
Department of Host-Pathogen Interaction - DHPI

THÈSE
présentée par :

Sultan ALI

soutenue le : 02 Avril en 2013

pour obtenir le grade de : Docteur de l’université de Strasbourg
Discipline / Spécialité : Science du Vivant / Aspects cellulaire et moléculaires de la
Biologie

Induction par Vpr de la dégradation de la protéine CTIP2 via la
voie du protéasome dans les cellules microgliales.

THÈSE dirigée par :
ROHR Olivier Professeur, université de strasbourg
SCHWARTZ Christian (Co-directeur) MCU HDR, université de Strasbourg
RAPPORTEURS :
MARGOTTIN-GOGUET Florence Dir. de recherches, Institut Cochin, Paris
EKSTRÖM Tomas J Pr. Karolinska Institut, Stockholm, Sweden.
VITALE Nicolas Directeur de recherches, UDS.
EXAMINATEUR :
BAUSERO Pedro Pr, Univ. Pierre et Marie Curie Paris.
Acknowledgments

I humbly bow my head before God, the most beneficent and merciful,
whose blessing flourished my thoughts and thrived my ambitions. I would
like to express my deep and sincere gratitude to my co-Director of the
research unit, Professor Ermanno Candolfi, for making it possible for me
to work in such a prestigious scientific environment.
I feel immense pleasure to gratefully acknowledge and to express deep
sense of gratitude to my supervisor, Pr. Olivier ROHR, for guiding me
throughout my course of study. Olivier has been very supportive,
encouraging and kind. I owe a great deal of appreciation for his valuable
advice, constructive criticism and valuable discussions around my work. It
has been a great honor to have him as a supervisor. Apart from his
scientific knowledge and devotion to research, I found him an adorable,
pure and kind-hearted human being.
I would like to express my deep and sincere gratitude to my co-
supervisor, Dr. Christian Schwartz, for his enthusiasm, constructive
discussions and motivation throughout the course of my stay under his
supervision.
I wish to express my cordial appreciation to my examiners, Dr. Florence
Margottin-Goguet, Pr. Tomas J Ekström, Dr. Nicolas Vitale and Pr. Pedro
Bausero for the acceptance to be a referee. I feel immense pleasure to
gratefully acknowledge and to express deep sense of gratitude to Dr.
Valentin le Douce for his enthusiasm, creative suggestions, motivation
and exemplary guidance throughout the course of my doctoral research
and also during thesis writing. Moreover, I offer my profound gratitude to
Thomas Cherrier for his scientific and moral support during my earlier
period of stay. Words are lacking to express my gratitude for Higher
Education Commission of Pakistan, for providing me with scholarship
during my study in France. The opportunity of studying in France has
really broadened my horizon and widened my perspectives in life.
I sincerely acknowledge my colleagues especially Raphael Riclet, Andrea
Janossy, Emeline Pelletier, Marion Pouget, Julie Brunet and my other
colleagues from the parasitology team for their support during my
projects and would like to extend my thanks to Esterina Hoffmann for
facilitating all administrative issues.
I own my sincere thanks to Rizwan Aslam, Ghulam Hussain, Asghar
Shabbir, M Nauman Zahid and Azeem Sultan for their support, guidance
and affection. I would also like to extend huge, warm thanks to my other
Pakistani fellows Sarfraz, Adnan, Niaz, Azhar, Rafiq and Ikram for their
love and sincerity. I am eternally grateful to my parents, Basharat Ali &
Sakina Bibi, my brother M Usman, my sisters, my beloved Rimsha Hassan
and other family members for their unconditional love, fidelity, endurance
and encouragement. There are so many others whom I may have
inadvertently left out and I sincerely thank all of them for their help.
Table of Contents
Introduction....................................................................... 1
1. Acquired immunodeficiency syndrome and HIV ........................ 2
1.1. Discovery of AIDS and HIV ............................................................ 2
1.2. Epidemiology ............................................................................... 3
1.3. Types of HIV ............................................................................... 5
1.4. Pathogenesis of AIDS ................................................................... 6
2. Description of HIV-1 and its life cycle ....................................... 8
2.1. Structure of infectious viral particle ................................................ 8
2.2. Viral genome ............................................................................. 10
2.3. Replication of HIV-1 ................................................................... 12
2.4. HIV-1 Infection cure ................................................................... 22
3. HIV accessory proteins ........................................................... 23
3.1. Negative regulatory factor (Nef) ................................................... 23
3.2. Viral protein unique (Vpu) ........................................................... 24
3.3. Viral infectivity factor (Vif) .......................................................... 25
3.4. Viral protein Regulatory (Vpr) ...................................................... 25
4. Ubiquitination ......................................................................... 31
4.1. Process of ubiquitination ............................................................. 32
4.2. Types of Ubiquitination ............................................................... 33
4.3. Components of ubiquitin ligase .................................................... 35
4.4. Effects of ubiquitination .............................................................. 36
5. Viruses using host ubiquitin system ....................................... 38
5.1. Adenoviruses ............................................................................. 39
5.2. Herpes viruses ........................................................................... 41
5.3. Papillomaviruses ........................................................................ 42
5.4. Poxviruses ................................................................................. 43
5.5. Parvoviruses .............................................................................. 44
5.6. Reoviruses ................................................................................ 44
5.7. Orthomyxoviruses ...................................................................... 45
5.9. Retroviruses .............................................................................. 45
6. Restriction factor .................................................................... 52
6.1. Hallmarks of restriction factors .................................................... 52
6.2. TRIM5 ....................................................................................... 54
6.3. Tetherin .................................................................................... 55
6.4. APOBEC3 .................................................................................. 57
6.5. SAMHD1 ................................................................................... 61
7. Reservoir and latency ............................................................. 65
7.1. Anatomical reservoirs ................................................................. 65
7.2. Cellular reservoirs ...................................................................... 65
7.3. Molecular latency ....................................................................... 68
7.4. CTIP2 ....................................................................................... 73
7.5. Vpr and Cul4A-DDB1DCAF1 E3 ubiquitin ligase .................................. 78
Aim of study ..................................................................... 83
Results ............................................................................. 86
Supplementary results ................................................... 107
Discussion ...................................................................... 122
Materials and methods ................................................... 130
Bibliography................................................................... 133
Annexes ......................................................................... 166
List of Figures
Figure 1: Estimated adults and children living with HIV in 2011. ...................... 3
Figure 2: Global HIV trends, 2001-11 ........................................................... 4
Figure 3: Financial plan for 2015to eradicate HIV. .......................................... 4
Figure 4: Time course of HIV-1 infection. ...................................................... 7
Figure 5A: Schematic overview of infectious virion of HIV-1 ........................... 9
Figure 6: Schematic representation of HIV-1 and HIV-2 integrated genomes ... 11
Figure 7: Schematic representation of life cycle of HIV ................................. 12
Figure 8: Schematic overview of HIV entry from attachment to fusion of the
membranes ............................................................................................ 14
Figure 9: Intracellular transport of HIV-1 and DNA Flap-dependent nuclear
import of PIC ........................................................................................... 16
Figure 10: HIV-1 genome transcription and splicing of mRNAs. ...................... 21
Figure 11: Schematic overview of HIV-1 assembly, release and maturation .... 21
Figure 12A/B: Vpr structure and its functions .............................................. 26
Figure 13: Ubiquitination and its types ........................................................ 34
Figure 14: Proteasomal degradation of poly-ubiquitinated protein by the 26S
proteasome ............................................................................................. 37
Figure 15: A schematic overview of examples of viral effects on host ubiquitin
system ................................................................................................... 51
Figure 17: Overview of Vpu-mediated tetherin regulation .............................. 57
Figure 22: Degradation of APOBEC3G by Vif by host ubiquitin proteasome
system .................................................................................................. 60
Figure 23: Effect of SAMHD1 on dNTP and Vpx can block this process ............. 62
Figure 24: Replication of reteroviruses in dendritic cell with Vpx and without Vpx
............................................................................................................. 64
Figure 16: Establishment of a latent reservoir in resting T-cell ....................... 66
Figure 17: Cells of monocyte/macrophage lineage ........................................ 67
Figure 18: Molecular structure of HIV-1 LTR ................................................ 70
Figure 19: Epigenetic modifications and control of transcription ..................... 72
Figure 25: CTIP2 represses the HIV-1 transcription by favoring heterochromatin
structure ................................................................................................ 76
Figure 26: CTIP2 can repress P-TEF-b target genes ..................................... 76
Figure 27: Direct & indirect effects of CTIP2 on HIV-1 transcription ................ 77
Figure 28: Summary of restriction factors and their counteraction in myeloid
cells ...................................................................................................... 81
List of Tables

Table 1: List of viruses using UPS and function modifications. ............................ 48
Table 2: Proteasome-associated proteins ...................................................... 115
Table 3: Upregulated proteins after CTIP2 overexpression in microglial cells. ..... 116
ABBREVIATIONS

AIDS: Acquired ImmunoDeficiency Syndrome
APOBEC: APOlipoprotein B mRNA-editing, Enzyme-Catalytic,
polypeptide-like
ATR: Ataxia Telangiectasia and Rad3-related protein
CDK9: Cyclin-dependent kinase 9
CT1: Cyclin T1
CTIP2: COUP-TF Interacting Protein 2
DCAF1: DDB1-Cul4A Adaptor Factor 1
DDB1: DNA Damage-Binding protein 1
dNTP: Deoxyribonucletide triphosphate
GALT: Gut-Associated Lymphoid Tissue
Gp160/120/41: GlycoProtein 160/120/41
H3K4me3: Tri-methylation of lysine 4 of Histone H3
H3K9me3: Tri-methylation of lysine 9 of Histone H3
HA: Hemagglutinin
HAART: Highly Active AntiRetroviral Therapy
HDAC: Histone DeACetylase
HEXIM: Hexamethylene bis-acetamide inducible 1
HIV: Human Immunodeficiency Virus
HMBA: Hexamethylene bis-acetamide
HMT: Histone MethylTransferase
HP1: Heterochromatin Protein 1
HPBP: Human Phosphate Binding Protein
LSD1: Lysine Specific Demethylase 1
LTNP: Long-Term Non-Progressor
LTR: Long Terminal Repeat
miRNA: micro RNA
NES: Nuclear Export Signal
NF-κB: Nuclear Factor κB
NLS: Nuclear Localization Signal
NUC-1: Nucleosome 1
PIC: Pre-Integration Complex
P-TEFb: Positive Transcription Elongation Factor-b
RTC: Reverse Transcription Complex
siRNA: Small Interfering RNA
SIV: Simian Immunodeficiency Virus
Sp1: Specificity protein 1
SUV39H1: SUppressor of Variegation 3-9 Homolog 1
TAR: Transactivator Response Element
TAT: TransActivator of Transcription
TRIM: Triparitite Motif Protein
TSA: TrichoStatine A
UNG2: Uracil DNA Glycosylase 2
UPS: Ubiquitin Proteasome System
Vpr: Viral Protein Regulatory
VSV: Vesicular Stomatitis Virus