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Table of contents
1 ALPHAVIRUSES
Classifications
Taxonomy of Alphavirus
Arbovirus: an ecological group
Viral particle structure and genome organization
Non-structural proteins
Structural proteins
Replicative cycle in mammals and mosquitos
Binding and entry by endocytosis
Fusion and Viral genome release
Replication complex and assembly
2 CHIKUNGUNYA VIRUS – AN OLD-WORLD ALPHAVIRUS
Discovery
Transmission cycles and vector distribution
Epidemiology
Infectious route in competent vectors
The initial site of infection: the midgut
The final site of infection: the salivary glands
Pathogenesis and cellular tropism in vertebrate hosts
Human infection: clinical signs
Acute and chronic phases
Vaccine, antiviral treatments, and strategies in humans and mosquitos
Vaccine and antiviral treatments in humans
Antiviral strategies in the mosquito
3 CELLULAR RESPONSES IN ALPHAVIRUSES-INFECTED MAMMALS AND INSECTS
Innate immune response
In mammals
In the mosquito
Apoptosis
In mammals
3.2.1.1 The apoptosis signaling pathway
3.2.1.2 Regulation of apoptosis during alphavirus infection
3.2.1.3 Apoptosis-autophagy balance and crosstalk with other pathways In insects
3.2.2.1 The apoptotic pathway in Drosophila and Aedes
3.2.2.2 The effect of apoptosis on arbovirus outcome
4 ROLE OF P53 AND P53 ISOFORMS DURING ARBOVIRUS INFECTION IN MAMMALS AND INSECTS
Human p53
Discovery of p53
The p73/p63 and p53 family
Human p53 and p53 isoforms
Structure of p53 and p53 isoforms
Stabilization of p53
A transcription factor
Drosophila and mosquito p53
Dmp53/Dp53 and p53 isoforms in Drosophila
Recent discovery of two p53 isoforms in Aedes
Control of p53 pathways during viral infection
Function of p53 during “non-arboviral” infections
Different roles of p53 during arboviral infections: DENV, WNV and ZIKV
Modulation of p53 transcriptional activity by p53 isoforms during viral infection
OBJECTIVES
EXPERIMENTAL REPORT
CHAPTER 1) OPPOSITE EFFECT OF P53 ON CHIKV INFECTION IN A HUMAN CELL LINE AND IN VIVO IN DROSOPHILA MELANOGASTER
1 OBJECTIVES
2 MATERIAL AND METHODS
Cell lines and viruses
Generation of TP53 CRISPR-mediated knockout LHCN-M2 cell line
sgRNA design
Cloning in lentiCRISPRv2 plasmid
Lentivirus production
Stable cell line generation
Verification of gene knock-out by Western blot
Infection of knockout cell lines with CHIKV
Transfection of plasmid Flag-RIG-I 2CARD
Flow cytometry analysis
Western blot
RNA extraction and RT-qPCR
Cell viability assays
Subcellular fractionation
In vivo Drosophila melanogaster
Detection of Wolbachia by PCR and tetracycline treatment
Fly injection
Survival curve
RNA extraction of whole fly
TCID50/mL
Statistical analysis
3 RESULTS
IN MAMMALS: INNATE IMMUNE ANTIVIRAL ACTIVITY OF P53 IN HUMAN SKELETAL MUSCLE CELLS INFECTED WITH CHIKV
Infection of an LHCN-M2 cell line by CHIKV induces stabilization of p53 protein
Infection of LHCN-M2 cell line by CHIKV induces Type I interferon immune signaling response
but not cell cycle arrest and not apoptotic p53 dependent
Effect of LHCN-M2 p53 deletion on CHIKV infection and cellular outcome
Generation of p53 knockout LHCN-M2 (sgRNA_p53) and luciferase (sgRNA_luc) control cell line using CRISPR/Cas9 technology
Infection of p53 knockout LHCN-M2 with CHIKV
Effect of p53 knockout on cell viability and p53-target genes during CHIKV infection
Effect of p53 knockout on interferon Type-I signaling during CHIKV infection
Capacity of p53 knockout LHCN-M2 cells to induce the Type-I interferon signaling pathway
Effect of p53 knockout on CHIKV-induced cell death
Nuclear translocation of p53 and NF-κB during CHIKV infection
Effect of p53 knockout on the release of cytochrome c during CHIKV infection
Discussion
IN INSECTS: IN DROSOPHILA MELANOGASTER P53 EXPRESSION IMPACTS THE VIRAL REPLICATION OF CHIKV AND SINV
Detection of Wolbachia spp. in Drosophila w1118 and p53-/- strains
Survival curve of w1118 and p53-/- flies injected with CHIKV
Viral replication of SINV and CHIKV and CHIKV production in w1118 and p53-/- strains
Discussion
IN MOSQUITO CELLS: INFECTION OF MOSQUITO AEDES ALBOPICTUS AND AEDES AEGYPTI CELL LINES WITH CHIKV
RESULTS AND DISCUSSION
Effect of the origin of CHIKV production on the permissiveness and pro-apoptotic response of
mosquito cell lines
Time course of CHIKV infection in C6/36 cells and analysis of pro-apoptotic and antioxidant
p53-target genes
Conclusion
CHAPTER 2) ANALYSIS OF THE EFFECT OF P53 ISOFORMS ON CHIKV INFECTION IN MAMMALIAN CELL LINES
1 OBJECTIVES
2 MATERIAL AND METHODS
Cell lines and viruses
Generation of TP53 CRISPR-mediated knockout LHCN-M2 and U2OS cell line
Transduction of LHCN-M2 with doxycycline-inducible shRNA-Δ40p53
Generation of Doxycycline-inducible system for overexpression of Δ40p53α and Δ133p53α isoforms
2.2.2.1 Production of VSVg pseudo particles
2.2.2.2 Verification of doxycycline-inducible system for overexpression of p53 isoforms in LHCN-M2 cells
Cell viability
Immunostaining and flow cytometry analysis
3 RESULTS
Generation of an endogenous Δ40p53 isoform overexpressing cell line using CRISPR/Cas9 technology
Effect of Δ40p53 overexpression on CHIKV infection
Change in cell morphology of the LHCN-M2 Δ40p53 cell line
Effect of Δ40p53 isoform in the new generated CRISPR/Cas9 LHCN-M2 cell line on the cell viability of LHCN-M2 during CHIKV infection
Loss of effect of the Δ40p53 isoform on viral infection in the CRISPR/Cas9 U2OS cell line
Doxycycline-inducible system overexpressing Δ40p53 and Δ133p53 isoforms
Generation of the LHCN-M2 doxycycline-inducible system for the over-expression of Δ40p53 and
Δ133p53 isoforms
Effect of overexpression of p53 isoforms on CHIKV viral capsid
Discussion
GENERAL CONCLUSION
REFERENCES
