The inducible antiviral immune response of Drosophila melanogaster [Elektronische Ressource] / vorgelegt von Cordula Kemp

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The inducible antiviral immuneresponse of Drosophila melanogaster.vorgelegt vonDiplom-Ingenieurin fur¨ Medizinische BiotechnologieCordula Kempaus Cottinghamvon der Fakultat III¨Institut fur¨ Biotechnologieder Technischen Universitat Berlin¨zur Erlangung des akademischen GradesDoktorin der Ingenieurwissenschaften- Dr.-Ing. -genehmigte DissertationPromotionsausschuss:Vorsitz: Prof. Dr. L. GarbeGutachter: Prof. Dr. R. LausterGutachter: Prof. J.-L. Imlerhter: Prof. Dr. J. KurreckTag der wissenschaftlichen Aussprache: 30. Juli 2010Berlin 2010B83MEINEN ELTERN.DANKE.This thesis is dedicated to my parents.Thank you for all your love and support.The research for this thesis was performed duringdecember 2006 to december 2009in the Institut de biologie moleculaire et cellulaire, Strasbourg, France.AcknowledgmentsI would like to thank the many people who have been involved with thisthesis.First of all, I would like to thank Prof. Jules Hoffmann and Prof.Jean-Marc Reichhart for accepting me in the laboratory and allowing me towork in such a scientifically stimulating environment.My sincere thank you to my supervisor Prof. Jean-Luc Imler for all of yoursupport and guidance whilst working in the lab and during the process ofwriting my thesis. I have learned so much during the time I spent in yourgroup.I would also like to thank as well my second supervisor Prof.

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The inducible antiviral immune
response of Drosophila melanogaster.
vorgelegt von
Diplom-Ingenieurin fur¨ Medizinische Biotechnologie
Cordula Kemp
aus Cottingham
von der Fakultat III¨
Institut fur¨ Biotechnologie
der Technischen Universitat Berlin¨
zur Erlangung des akademischen Grades
Doktorin der Ingenieurwissenschaften
- Dr.-Ing. -
genehmigte Dissertation
Promotionsausschuss:
Vorsitz: Prof. Dr. L. Garbe
Gutachter: Prof. Dr. R. Lauster
Gutachter: Prof. J.-L. Imlerhter: Prof. Dr. J. Kurreck
Tag der wissenschaftlichen Aussprache: 30. Juli 2010
Berlin 2010
B83MEINEN ELTERN.
DANKE.
This thesis is dedicated to my parents.
Thank you for all your love and support.
The research for this thesis was performed during
december 2006 to december 2009
in the Institut de biologie moleculaire et cellulaire, Strasbourg, France.Acknowledgments
I would like to thank the many people who have been involved with this
thesis.
First of all, I would like to thank Prof. Jules Hoffmann and Prof.
Jean-Marc Reichhart for accepting me in the laboratory and allowing me to
work in such a scientifically stimulating environment.
My sincere thank you to my supervisor Prof. Jean-Luc Imler for all of your
support and guidance whilst working in the lab and during the process of
writing my thesis. I have learned so much during the time I spent in your
group.
I would also like to thank as well my second supervisor Prof. Roland
Lauster who I started my scientific career with and who always had an
open ear for me.
Thanks to everyone who participated to this work. I want to address a
special thank you to all the members of the group and the institute that
have shared this time of my life with me and became much missed friends.
I would like to especially thank Safia, Steffi M., Ioannis, Stan, Yann, Steffi
L. and Basti. There is not enough room to thank you for everything you
did, let’s simply say, this thesis would not have been finished without you. I
wish you all the best for the future.
A big thank you to Estelle and Miriam for all your technical support. I
equally would like to say thank you to Laurent Troxler and Charles Hetru
for their bioinformatic support. I would like to thank as well the best
student I ever had the pleasure to supervise, Francois and the recent team
members Karim and Jessica. I wish you lots of luck for your projects.
A last but not least very special thank you to my wonderful husband and
family that supported me all the way through. Thank you!Abbreviations
aa amino acids
AaSTAT Aedes albopictus STAT
ACTH adrenocorticotrophic hormone
AGO Argonaute
AMP antimicrobial peptide
ANV American Nodavirus
bp base pair
CARD caspase recruitment domain
CARDIF CARD adapter inducing IFN-β
cDNA complementary DNA
cds coding sequence
CGM complete growth medium
CFM Chifoumi
CrPV Cricket paralysis virus
DAP meso-diaminopimelic acid
DBV Drosophila Birnavirus
DC dendritic cell
DCL Dicer-like
DCV Drosophila C virus
DNA desoxy ribonucleid acid
DREDD Death related ced-3/Nedd2-like
dsRNA double stranded RNA
DTV Drosophila Totivirus
DXV X virus
eIF2 elongation initiation factor 2
ER endoplasmatic reticulum
EST expressed sequence tag
FADD Fas-associated Death Domain
FHV Flockhouse virus
GBP-1 guanylate binding protein-1
GNBP Gram-negative binding protein
GPCR G-protein-coupled receptors
GTPase guanosine triphosphatase
h hours
HBD human β-defensin
HSPG heparan sulphate proteoglycan
IFN interferon
IIV 6 Invertebrate iridescent virus 6
imd immune deficiencyIPS-1 IFN-β promotor stimulator-1
IRC immune responsive catalase
IRES internal ribosomal entry site
IRF interferon regulatory factor
ISG IFN-stimulated gene
ISRE response element
Jak Janus kinase
JEV Japanese encephalitis virus
LDLR low-density-lipoprotein receptor
MAP3K Mitogen-Activated Protein 3 kinase
MAVS mitochondrial antiviral signaling
MDA-5 melanoma differentiation-associated gene 5
μg microgram
min minute
ml milliliter
Mx myxovirus resistance
NCBI National Center for Biotechnology Information
ng nanogram
nt nucleotide
NLR NOD-like receptor
OAS 2’-5’ oligo adenylate synthetase
PAMP pathogen associated molecular pattern
PCR polymerase chain reaction
PGN peptidoglycan
PGRP p recognition protein
PKC protein kinase C
PRR pattern recognition receptor
Q-PCR quantitative real time PCR
RC2 retrocyclin 2
RdRP RNA dependent RNA polymerase
RIG-I retinoic acid-inducible gene I
RLR RIG-I-like receptor
RNA ribonucleic acid
RNAi RNA interference
ROS reactive oxygen species
RIP receptor interacting protein
rpm rounds per minute
SIGMAV sigma virus
SH2 Src homology 2
SINV Sindbis virus
siRNA small interfering RNA
STAT signal transducers and ativators of transcriptionTBK1 TANK binding kinase 1
TCT tracheal cytotoxin
TIR Toll/IL-1 receptor
TLR Toll-like recptor
TNF tumor necrosis factor
TotA Turandot A
TYK2 Tyrosine kinase 2
upd unpaired
vRDRP viral RNA dependent RNA polymerase
VAMP virus associated molecular pattern
vir-1 virus-induced RNA1
VISA signaling adapter
VSR viral suppressors of RNAi
VSV vesicular stomatis virusZusammenfassung
In der vorliegenden Arbeit wurde Drosophila melanogaster als Modell ge-
nutzt, um die angeborene Immunantwort gegen virale Infektionen zu studie-
ren.
Wir untersuchten mit Hilfe von genomweiten microarrays das Transkriptom
von Fliegen, welche entweder mit dem Drosophila C Virus (DCV), dem Flock-
house Virus (FHV) oder dem Sindbis Virus (SINV) infiziert waren. Infektion
mit diesen drei positiv orientierten Einzelstrang RNS Viren fuhrte zu einer¨
starken transkriptionellen Antwort, welche deutlich virusspezifische Indukti-
onsmuster zeigte. Um die biologische Signifikanz dieser beobachteten Muster
zu studieren, testeten wir Mutanten verschiedener Signalwege, namlic¨ h den
Imd, den Toll und den Jak-STAT Signalweg, fur Suszebtibiltat nach viraler¨ ¨
Infektion. Wir fuhrten¨ eine Vergleichsstudie durch, in welcher wir ein Set von
5 verschiedenen RNS Viren benutzten. Das Set beinhaltete die positiv ori-
entierten RNS Viren DCV, Cricket Paralysis Virus (CrPV), FHV und SINV
und den negativ orientierten RNS Virus Vesicular Stomatis Virus (VSV).
Die Dicistroviren DCV und CrPV formten eine spezielle Gruppe unter den
getesteten Viren, da Mutanten fur¨ den Jak-STAT und den Toll Signalweg
anfallig fuer DCV und CrPV Infektion waren. Mutanten starben fruher als¨ ¨
Wildtyp Fliegen und zeigten erhoh¨ te virale Titer. Im Gegensatz dazu, sa-
¨hen wir keinen Phanotyp, weder im Uberleben noch in den viralen Titern¨
nach Infektion mit FHV, SINV oder VSV. Der Imd Signalweg wurde nicht
als eine bedeutende Komponente der antiviralen Immunantwort identifiziert.
Trotzdem zeigten Mutanten dieses Signalweges eine Tendenz zu reduzierten
viralen Titern nach Dicistrovirus Infektion. Wir zeigten weiterhin, dass re-
lativ gesehen, der Jak-STAT Signalweg genauso bedeutend fur die Abwehr¨
gegen Dicistriviren ist wie RNS Interferenz. Diese Daten unterstreichen die
Wichtigkeit der induzierbaren Antwort in Drosophila.
Wir charakterisierten Chifoumi (cfm) alias CG11501 als ein Beispiel fuer
ein virusinduziertes Gen. Wir zeigten, dass cfm ein zirkulierendes Peptid ko-
diert, welches nach viraler Infektion im Fettkorp¨ er exprimiert und in den He-
molymph sekretiert wird. Mutanten fur dieses Gen, zeigten reduzierte virale¨
¨Titer und verl¨angertes Uberleben nach Infektion mit DCV. Diese Ergebnisse
legen Nahe, dass CFM m¨oglicherweise ein wirtskodierter Faktor ist, welcher
die virale Replikation in Drosophila fordert.¨Abstract
The work presented in this thesis used the fruit fly Drosophila melanogaster
as a model system to study the inducible innate immune response against
virus infection.
We studied the transcriptome of flies infected with either Drosophila C virus
(DCV), Flockhouse virus (FHV) or Sindbis virus (SINV) using genome-wide
microarray analysis. Infection with these postive-sense single-stranded RNA
viruses triggered a strong inducible response that showed evidence of virus-
specific induction patterns. In order to study the biological significance of
these gene inductions, we investigated susceptibility to viral infection of mu-
tant flies for signaling pathways, such as the Imd, the Toll and the Jak-STAT
pathway. We conducted a comparative analysis using a set of five distinct
RNA viruses, that included the positive-sense RNA viruses DCV, Cricket
paralysis virus (CrPV), FHV and SINV, and the negative-sense RNA virus
Vesicular stomatis virus (VSV). We found that the Dicistroviruses DCV and
CrPV formed a unique group among the viruses tested, as mutants of both,
the Jak-STAT and the Toll pathway were susceptible to infection with DCV
and CrPV. The observed susceptibility correlated with an increase in viral
titers. No phenotype, neither in viral load nor in survival susceptibility was
observed upon infection with FHV, SINV or VSV. We found no major role
for the Imd pathway in the antiviral defense, however, mutant flies of this
pathway showed a trend towards a reduction in Dicistrovirus load. Further-
more, we showed that the Jak-STAT pathway is as important for the defense
against Dicistrovirus infection as RNA interference, highlighting the relative
importance of the inducible response against this virus family.
As an example for a virus-induced gene, we characterized Chifoumi (cfm)
alias CG11501. We showed that cfm codes for a circulating peptide that is
induced in the fat body upon viral infection and secreted in the hemolymph.
cfm mutant flies showed decreased viral titers and prolonged survival upon
infection with DCV, suggesting that CFM may function as a host-encoded
factor that propagates viral replication in drosophila.Contents
1 Introduction: Antibacterial and antifungal innate immunity
in drosophila 3
1.1 Epithelial and cellular defense in Drosohila ........... 3
1.2 The humoral response ...................... 5
1.2.1 Antimicrobial effectors .................. 5
1.2.2 Microbial recognition ................... 6
1.3 Signaling pathways implicated in innate immunity of drosophila 8
1.3.1 The Toll pathway ..................... 8
1.3.2 The Imd pahway 9
1.3.3 The Jak-STAT pathway .................1
1.4 Conserved aspects of drosophila innate immunity .......17
1.4.1 Toll and TLRs: lessons from drosophila.........17
1.4.2 NF-κB and apoptosis: The TNF-Imd connection....19
2 Introduction: Innate antiviral defense strategies in plants
and mammals 21
2.1 Plant antiviral defense ......................21
2.2 Mammalian innate antiviral immunity .............2
2.2.1 Viral recognition .....................2
2.2.2 The IFN system27
3 Introduction: Antiviral immunity of D. melanogaster 33
3.1 Models of Drosophila viral infection ...............33
3.1.1 Drosophila viruses ....................34
3.1.2 Non-drosophila viruses ..................36
3.1.3 Insect DNA viruses36
3.2 RNA interference: A sequence-specific antiviral defense ....37
3.2.1 Biogenesis and functions of small RNAs ........37
3.2.2 Small interfering RNAs: Tiny helpers against viral in-
fection ...........................39
3.3 The inducible response ......................43
iCONTENTS ii
3.3.1 The Toll and Imd pathway................43
3.3.2 The Jak-STAT pathway .................4
3.3.3 Dicer-2 mediated inducible antiviral activity ......46
3.3.4 Autophagy ........................47
3.4 Importance of studying virus-insect interaction.........47
3.4.1 Economical aspects ....................48
3.4.2 Insect disease vectors ...................48
3.4.3 Aims of this thesis49
4 Material and Methods 52
4.1 Fly strains .............................52
4.2 Infections and survival ......................52
4.3 Cell culture ............................53
4.4 DNA extraction ..........................53
4.5 Plaque assay53
4.6 TCID50 ..............................54
4.7 RNA analysis ...........................5
4.7.1 RNA extraction5
4.7.2 cDNA synthesis5
4.7.3 Semiquantitative reverse transcriptase
PCR............................56
4.7.4 Quantitative real time PCR ...............56
4.7.5 Microarray analysis ....................58
4.7.6 In situ hybridisation ...................58
4.8 Protein analysis ..........................62
4.8.1 Sample preparation and protein Quantification.....62
4.8.2 SDS-PAGE ........................62
4.8.3 Western Blot .......................63
4.9 Generation of CFM specific antiserum .............63
4.10 Statistical analysis63
4.11 Cell based Jak-STAT reporter assay...............64
4.11.1 Preparation of dsRNA ..................64
4.11.2 Transfection64
4.12 In vitro assays using recombinant Chifoumi...........65
4.12.1 Antibacterial assays....................65
4.12.2 Pretreatment of viruses with CFM65
4.12.3 Cytokine assay ......................6
5 Results: A comparative study of the drosophila immune re-
sponse towards infection with RNA viruses 68
5.1 RNA virus triggers a complex transcriptional response 69