Mechanism and consequences of interferon γ inhibition by the viral interferon regulatory factor 3 (vIRF-3) of Kaposi’s sarcoma-associated herpesvirus (KSHV) [Elektronische Ressource] = Mechanismus und Auswirkungen der Inhibierung von Interferon γ durch den viralen Interferon-regulatorischen Faktor 3 (vIRF-3) des Kaposi Sarkom-assoziierten Herpesvirus (KSHV) / Anna Katharina Schmidt. Betreuer: Bernhard Fleckenstein

De
Mechanism and consequences of interferon γ inhibition by the viral interferon regulatory factor 3 (vIRF-3) of Kaposi’s sarcoma-associated herpesvirus (KSHV) Mechanismus und Auswirkungen der Inhibierung von Interferon γ durch den viralen Interferon-regulatorischen Faktor 3 (vIRF-3) des Kaposi Sarkom-assoziierten Herpesvirus (KSHV) Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Anna Katharina Schmidt aus Erlangen Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 15.06.2011 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatter: Prof. Dr. Bernhard Fleckenstein Zweitberichterstatter: Prof. Dr. Robert Slany Mechanism and consequences of interferon γ inhibition by the viral interferon regulatory factor 3 (vIRF-3) of Kaposi’s sarcoma-associated herpesvirus (KSHV) Contents 1 Summary________________________________________________________5 2 Zusammenfassung ________________________________________________6 3 Introduction _____________________________________________________7 3.1 Kaposi’s sarcoma-associated herpesvirus______________________________ 7 3.
Publié le : samedi 1 janvier 2011
Lecture(s) : 24
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Source : D-NB.INFO/1015475272/34
Nombre de pages : 93
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Mechanism and consequences of interferonγinhibition
by the viral interferon regulatory factor 3 (vIRF-3)
of Kaposis sarcoma-associated herpesvirus (KSHV)
Mechanismus und Auswirkungen der Inhibierung von Interferonγ
durch den viralen Interferon-regulatorischen Faktor 3 (vIRF-3)
des Kaposi Sarkom-assoziierten Herpesvirus (KSHV)
Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von Anna Katharina Schmidt aus Erlangen
Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg dlichen Prüfung: 15.06.2011
Prof. Dr. Rainer Fink
Vorsitzender der Promotionskommission:
Prof. Dr. Bernhard Fleckenstein
Prof. Dr. Robert Slany
Tag der mün
Zweitberichterstatter:
Erstberichterstatter:
4
oj _________________________________________________Pr ect rationale 15
3
3.4Antigen presentation and MHC class II ______________________________ 14
3.3
3.2
3.1
_____________________________________________________ Introduction 7
vIRF-3 suppresses CIITA transcription _ _____ 23________________________
vIRF-3 regulates MHC II expression ________________________________ 19
Microarray analysis revealed MHC II as potential target of vIRF-3_______ 16
5
Results 16_________________________________________________________
rferonγ_____________________________________________________ Inte 13
Interferon regulatory factors ________________________________________ 8
Kaposis sarcoma-associated herpesvirus 7______________________________
Contents
of Kaposis sarcoma-associated herpesvirus (KSHV)
Mechanism and consequences of interferonγinhibition
by the viral interferon regulatory factor 3 (vIRF-3)
Zusammenfassung _____________________________________ 6___________
2
5
Summary ________________________________________ ________________
1
vIRF-3 regulates IFNγ____________________________________________ 27
______________________________ Cellular IRF5 and IRF7 bind to vIRF-3 34
IRF5 and IRF7 activate theIFNγpromoter __________________________ 35_
vIRF-3 inhibits IRF5 binding and activation of theIFNγ 39promoter _______
IRF5 binds to the proximal part of theIFNγpromoter ______ ___________
41
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
6
______________________________________________________ Discussion
6.1
6.2
Role of vIRF-3 in the immune evasion of KSHV _______________________
Role of IRF5 in the immune response _____________________ ___________
7Material and Methods ____________________________________________
8
9
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
_____________________________________________________ Cell culture
_____________________________________________________ Transfection
46
46
50
54
54
55
___________________________________________________ DNA methods 58
Protein methods 60__________________________________________________
___________________________________________________ RNA methods 64
DNA-Protein Interaction 66__________________________________________
Lucif y__________________________________________________ erase assa 69
Bioinformatics and statistics 69_______________________________________
Ref _____________________________________________________ erences 70
Appendix ________________________________________________80_______
1 Summary
Summary
Kaposi sarcoma associated herpesvirus (KSHV), also termed human herpesvirus-8
(HHV-8), is the causative agent of Kaposis sarcoma and of two lymphoproliferative
disorders, multicentric Castleman disease and primary effusion lymphoma (PEL). The
viral interferon regulatory factor 3 (vIRF-3) is required for the continuous
proliferation of cultured PEL cells and thus, a potential oncogene of KSHV. Besides
regulation of proliferation, vIRF-3 also modulates the immune response to KSHV-
infected cells. In this study, inhibition of interferonγ (IFNγ) synthesis and thereby
inhibition of antigen presentation were identified as new functions of vIRF-3.
Furthermore, a mechanism of IFNγ by vIRF-3 was explained; the cellular inhibition
interferon regulatory factor 5, which is counteracted by vIRF-3, was identified as a
regulator ofIFNγtranscription. Regulation of major histocompatibility complex class
II (MHC II) transcription by vIRF-3 was identified in microarrays. Knockdown of
vIRF-3 led to increased MHC II levels in KSHV-infected PEL cells, whereas
overexpression of vIRF-3 resulted in downmodulation of MHC II in KSHV-negative
B cells. MHC II regulation by vIRF-3 could be traced back to repression of the MHC
II master switch regulator, class II transactivator (CIITA). Both the IFNγ-sensitive
CIITApromoter PIV and the B cell specific promoter PIII were inhibited by vIRF-3.
Consistently, IFNγlevels increased upon vIRF-3 knockdown in PEL cells, and vIRF-
3 was able to repressIFNγ promoter reporters. Examination of the cellular binding
partners of vIRF-3 - IRF3, IRF5 and IRF7 - on their influence onIFNγtrnoicsnatpir
revealed that counteraction of IRF5 was responsible for IFNγ repression by vIRF-3.
IRF5 bound and activated theIFNγpromoter and both mechanisms were counteracted
by vIRF3. A binding site for IRF5 was narrowed down to a region between 262-462
bp upstream of the IFNγtranscription start. An activator protein 1 (AP-1) element was
important for IRF5 binding and activation.Thus, it can be concluded that counteraction of cellular IRF5 by vIRF-3 inhibits IFNγ and thereby secretion
decreases MHC II expression. These data suggest a novel mechanism of KSHV-
mediated immune evasion. Inhibition of antigen presentation by infected B cells would result in reduced immune surveillance by CD4+T cells. Furthermore, the data
show a role of the autoimmune disease associated IRF5 in the regulation of the
immune interferon IFNγ.
5
2 
Zusammenfassung
Zusammenfassung
Das Kaposi-Sarkom assoziierte Herpesvirus (KSHV), auch als humanes Herpesvirus Typ 8 (HHV-8) bezeichnet, kann neben dem Kaposi-Sarkom auch zwei lymphoproliferative Erkrankungen auslösen, die multizentrische Castleman Krankheit und das primäre Effusionslymphom (PEL). Der virale Interferon-regulatorische Faktor 3 (vIRF-3) ist essentiell für das kontinuierliche Wachstum von kultivierten PEL Zellen und gilt daher als potentielles Onkogen von KSHV. Neben seiner proliferationsfördernden Wirkung beeinflusst vIRF-3 auch die Immunantwort gegen KSHV infizierte Zellen. In dieser Arbeit wurden neue Funktionen von vIRF-3 in der Inhibierung der Interferonγ (IFNγ) Synthese und damit in der Reduktion der Antigenpräsentation identifiziert. Des weiteren wurde ein Mechanismus der IFNγ-Inhibition durch vIRF-3 gezeigt. Der zelluläre Interferon-regulatorische Faktor 5 (IRF5), der von vIRF-3 inhibiert wird, wurde hierbei als Aktivator der IFNγ-Transkription beschrieben. Durch Microarray-Analysen wurden MHC II Moleküle als potentielle Kandidaten für die Regulation durch vIRF-3 identifiziert. Das Ausschalten von vIRF-3 führte zu erhöhter MHC-II Präsentation in KSHV-positiven PEL Zellen, wohingegen die Überexpression von vIRF-3 die MHC-II Expression in KSHV-negativen B-Zellen erniedrigte. Die MHC-II Regulation durch vIRF-3 konnte auf die Regulation des Hauptschalters der MHC-II Synthese, den Klasse II Transaktivator (CIITA), zurückgeführt werden. Sowohl der IFNγ-sensitiveCIITA-Promoter PIV als auch der B-Zell-spezifische Promotor PIII wurden durch vIRF-3 inhibiert. Passend dazu stieg auch die Sekretion von IFNγ nach dem Ausschalten von vIRF-3 in PEL Zellen. Außerdem reprimierte vIRF-3 denIFNγ-Promotor in Reporterversuchen. Die Untersuchung des Einflusses zellulärer Bindungspartner von vIRF-3 -IRF3, IRF5 und IRF7 - auf dieIFNγ-Transkription zeigte, dass die Repression von IFNγ die durch Bindung von vIRF-3 an IRF5 vermittelt wird. IRF5 zeigte eine starke Bindung und Aktivierung desIFNγPromotors, die durch vIRF-3 inhibiert werden konnte. Die Bindestelle für IRF5 amIFNγin einer Region zwischen 262 und 462-Promoter wurde bp oberhalb desIFNγ-Transkritionsstarts lokalisiert. Ein AP-1 Motiv war wichtig für die Aktivierung desIFNγ-Promoters durch IRF5. Zusammenfassend wurde gezeigt, dass vIRF-3 durch Inhibition des zellulären IRF5 die IFNγ hemmt. Durch Sekretion diesen Mechanismus führt vIRF-3 zu einer verminderten MHC-II Präsentation auf PEL Zellen, was einen neuen Mechanismus der Immunevasion durch KSHV darstellt. Des weiteren wurde eine Rolle für den mit vielen Autoimmunerkrankungen assoziierten IRF5 in der Regulation von IFNγentdeckt.
6
3 
Introduction
Introduction
3.1 Kaposis sarcoma-associated herpesvirus
Kaposis sarcoma-associated herpesvirus (KSHV), also termed human herpesvirus 8
(HHV-8), was discovered in 1994 by representational difference analysis of biopsies of Kaposis sarcoma, an endothelial skin cancer1. KSHV is found in all four epidemiological forms of Kaposis sarcoma (KS), (1) the classic KS, which affects
elderly men of Mediterranean or eastern European Jewish ancestry, (2) the endemic
form of KS, frequent in parts of central and eastern Africa, (3) the iatrogenic KS, that
develops after immune suppression and (4) the epidemic form, also called AIDS-KS, that arose as a consequence of the AIDS epidemic2. Furthermore, KSHV is
associated with two kinds of B cell lymphoma, the primary effusion lymphoma
(PEL), which is a highly malignant non-Hodgkin lymphoma originating from post germinal center B cells3and the multicentric Castleman disease4. The prevalence of KSHV is below 10% in western countries5, but raises up to 90% in sub-Saharan Africa6;7. KSHV is transmitted in regions with high seroprevalence mainly vertical
from mother to child, in other regions probably via saliva or among homosexual 8 partners .
After primary infection, KSHV establishes a life long persistence in the host, where it
changes between the two programs of latent and lytic infection. During latency, the
virus persists as a multicopy, circular, episomal DNA in the nucleus and expresses
only a few proteins that maintain the latent infection. The induction of lytic
replication results in expression of the entire set of viral genes, leading to the production of infectious viral progeny9.
KSHV belongs to theγ2-herpesvirus subgroup. Its closest relatives are the monkey viruses herpesvirus saimiri (HVS) and rhesus rhadinovirus (RRV); the nearest human pathogenic relative is theγ1-herpesvirus Epstein Bar virus (EBV)10. The double stranded DNA genomeof KSHV consist of around 165 kb and contains more than 80 open reading frames (ORFs)11ORFs of KSHV are homologous to HVS,. Most of the
but some  the so called K genes  are not shared with HVS. These K genes rather
show homology to cellular human genes and are therefore supposed to have been pirated by the virus during evolution12. Among them is a cluster of fourgenes with
7
Introduction
homology to cellular interferon regulatory factors, the viral interferon regulatory
factors vIRF-1 to vIRF-4.
3.2 Interferon regulatory factors
Cellular interferon regulatory factors Interferon regulatory factors (IRFs) were first characterized as transcriptional
regulators of the type I interferons (IFN) IFNαand IFNβand of IFN-inducible genes.
Beyond their role in early cellular response to pathogens, IRFs are important modulators of immune responses and hematopoietic differentiation13. In humans,
nine different members of the IRF family are known: IRF1, IRF2, IRF3, IRF4/
multiple myeloma oncogene 1 (MUM1), IRF5, IRF6, IRF7, IRF8/ interferon
consensus sequence binding protein (ICSBP), and IRF9/ interferon stimulated gene
factor 3γ (ISGF3γshare a common structure composed of an N-terminal). All IRFs
DNA binding domain (DBD) and a C-terminal IRF association domain (IAD). The
roughly 120 amino acid long DBD consists of a helix-turn-helix motif with five
conserved tryptophan residues, that mediate binding to IFN-stimulated response elements (ISRE, consensus sequence:A/GNGAAANNGAAACT) in responsive promoters14. The less conserved IAD mediates interaction with other IRFs for the formation of homo- and heterodimers or interacts with other transcription factors15.
IRFs can be classified according to different criteria: (1) by their transactivation
potential in transcriptional activators (IRF3, 5, 6, 7, 9) and in IRFs that can either
activate or repress gene transcription, depending on bound cofactors (IRF1, 2, 4, 8);
or (2) by their function, in regulators of immune cell function (IRF1, 2, 3, 5, 7, 9),
developmental regulators (IRF1, 2, 4, 8) or regulators of cell growth, either with oncogenic (IRF2, IRF4) or tumorsuppressive potential (IRF1, 5)13.
16;17 As immune regulators, IRF3 and IRF7 are the main inducers of type I interferon .
IRF3 is constitutively expressed in various cell types, IRF7 is constitutively expressed
in lymphoid tissue only, but its transcription is enhanced by interferon after infection
in various cell types. When a viral infection is recognized by toll-like receptors
(TLRs), IRF3 and IRF7 are phosphorylated at serine/threonine residues in the C -
terminal part by IκB Kinaseε (IKKε) and TANK binding Kinase 1 (TBK-1). This
leads to a dimerization and translocation into the nucleus where the IRFs interact with
8
Introduction
CREB binding protein (CBP)/p300 to activate the transcription of the type I
interferons IFNαand IFNβ.
IRF5 The IRF5 gene is encoded on chromosome 7q32 18. At least twelve different
transcripts of IRF5 have been characterized. They rise from combinations of (1) four different untranslated first exons19, (2) two alternative splice sites in the sixth exon20and (3) two different poly-A sites21. This results in eleven different protein isoforms,
because V4 and V5 differ only in the untranslated region; variants V9, V11 and V12 are truncated versions of IRF522. KSHV positive PEL cell lines express the IRF5 isoforms V1, V2, V3/4 and V523.
IRF5 is mainly expressed in lymphoid organs, especially B cells and plasmacytoid
dendritic cells (pDCs), but low levels of IRF5 are also detectable in other organs.
Additionally, IRF5 is inducible by IFNαin various cell types.
IRF5 resembles the common IRF protein structure, with a N-terminal DBD and a C-
terminal IAD (Figure 1A); additionally, an internal PEST domain that affects protein stability and a potentially autoinhibitory domain have been described24. IRF5 possesses a constitutive nuclear localization signal (NLS) and a stronger nuclear export signal (NES), so that IRF5 levels in the nucleus are normally low25; after phosphorylation of IRF5, an additional NLS is exposed and IRF5 is translocated to 24 the nucleus .
IRF5 is activated via TLR pathways 4, 7/8 and 9 (Figure 1B). After TLR stimulation,
IRF5 binds to the adapter complex containing myeloid differentiation primary-
response protein 88 (MyD88), IL-1 receptorassociated kinases 1 and 4 (IRAK-1/4) and TNF receptorassociated factor 6 (TRAF6)26. In this complex, IRF5 is K63 ubiquitinylated by the E3 ubiquitin ligase TRAF6 at lysine 410/41127. This ubiquitinylation is required for nuclear translocation27. Furthermore, IRF5 is phosphorylated by IKKα28, IKKε TBK-1 and25; phosphorylation by IKKα inhibits
TRAF6 mediated ubiquitinylation, whereas the phosphorylation by IKKεand TBK-1 seems to be necessary for dimerization29. Phosphorylated nuclear IRF5 can bind to
DNA as homodimer or as heterodimer with IRF3 or IRF7. Thereby, activation of
9
Introduction
IFNα is strongest for IRF5/IRF3 heterodimers, weaker for IRF5 homodimers24 and heterodimers of IRF5 with IRF7 inhibit IFNαproduction30 .
Figure 1: Structure, activation and functions of IRF5
A: The structure of IRF5 consists of an N-terminal DNA-binding domain (DBD) and a C-terminal IRF association domain (IAD); additionally, the nuclear export signal (NES), the two nuclear localization signals (NLS), the region where the variants differ in an insertion/deletion (In/Del) and the autoinhibitory domain (A) are depicted. B: IRF5 can be activated due to DNA damage and FasL signaling and due to viral infection via different TLR pathways. After ubiquitinylation and phosphorylation, IRF5 dimers translocate to the nucleus and activate transcription of tumorsuppressive and immune modulatory genes.
The target genes of IRF5 are not limited to type I interferons. Inflammatory cytokines like IL-6, TNFαand IL-12 are also induced by IRF531. Hence, IRF5 knockout mice
are resistant to lethal shock induced by CpG mediated upregulation of inflammatory cytokines32. While Takaokaet al. claim that IRF5 is dispensable for IFNαproduction32, other groups have shown that IRF5 knockout mice display lower levels of type I IFN in serum and macrophages after infection33 that IRF5 is essential and for type I IFN induction after TLR7 and 9 stimulation in dendritic cells34.
10
Introduction
In addition to its immune regulatory function, IRF5 acts as a tumor suppressor. It has been shown that IRF5 can be induced by p53 in response to DNA damage35. On the other hand, IRF5 sensitizes also p53-deficient cells to apoptosis after DNA damage36. Overexpression of IRF5 results in G2/M arrest and p53 independent apoptosis37.
Furthermore, IRF5 has been shown to be necessary for FasL-induced apoptosis in hepatocytes38.
Viral interferon regulatory factors KSHV encodes a cluster of fourgenes with homology to cellular interferon regulatory
factors. These viral interferon regulatory factors (vIRF-1 to vIRF-4) play important roles in the immune evasion of KSHV39. vIRFs exhibit significant homology to the DBD of the cellular IRFs, however they lack several of the conserved tryptophane residues and may therefore no longer be able to bind to DNA40;41. Roles in immune
modulation and in cell cycle regulation have been described for all vIRFs.
vIRF1 (K9) is a lytically expressed KSHV gene that acts as a negative regulator of interferon signaling42. It prevents the formation of the CBP/p300-IRF3 complex43;44, and inhibits p300 histone acetyltransferase activity by binding to p30045. Thereby, it
blocks the IRF3-mediated transcriptional activation of IFNα. In contrast, vIRF-1 does
not block the transactivation activity of IRF7, although it interacts weakly with IRF7. Moreover, there are potential oncogenic functions of vIRF-146;47. It has been shown to interact with p534849, ataxia telangiectasia mutated gene (ATM)50, Bcl2 interacting protein (BIM)51and the gene associated with retinoid-interferon-induced mortality-19 (GRIM19)52. In addition, vIRF-1 inhibits transforming growth factor ß (TGF-ß) mediated signaling via targeting of SMAD proteins53.
The KSHV protein vIRF-2 (K11.1) also counteracts the IFN system. It inhibits the
expression of the IFN-inducible genes driven by IRF1, IRF3, and ISGF3, but not by IRF754. Furthermore, vIRF-2 accelerates caspase mediated decay of IRF355.
Potential oncogenic functions of vIRF-2 comprise the inhibition of apoptosis by transcriptionalrepression of CD95 signaling56.
The viral interferon regulatory factor 3 (vIRF-3), also termed latency-associated
nuclear antigen 2 (LANA-2) or K10.5, is among the few viral genes expressed in all latently infected PEL cells57-60. vIRF-3 has been shown to be required for the
continuous proliferation of PEL cells in culture and can therefore be seen as an 11
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