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

Kaschmid

Description

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.

Sujets

• Gesundheit

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2011
• Nombre de lectures: 24
• Poids de l'ouvrage: 3 Mo

Extrait

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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

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durch den viralen Interferon-regulatorischen Faktor 3 (vIRF-3)

des Kaposi Sarkom-assoziierten Herpesvirus (KSHV)

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Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. 

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vorgelegt von Anna Katharina Schmidt aus Erlangen 

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          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:

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Erstberichterstatter:

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oj _________________________________________________Pr ect rationale 15

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3.4  Antigen 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

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of Kaposis sarcoma-associated herpesvirus (KSHV)

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Mechanism and consequences of interferonγinhibition

by the viral interferon regulatory factor 3 (vIRF-3)

Zusammenfassung _____________________________________ 6___________

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Summary ________________________________________ ________________

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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 

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______________________________________________________ Discussion

6.1 

6.2 

Role of vIRF-3 in the immune evasion of KSHV _______________________

Role of IRF5 in the immune response _____________________ ___________

7Material and Methods ____________________________________________

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7.1 

7.2 

7.3 

7.4 

7.5 

7.6 

7.7 

7.8 

_____________________________________________________ Cell culture

_____________________________________________________ Transfection

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___________________________________________________ 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_______

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1 Summary

Summary

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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 ofIFNγtranscription. 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 onIFNγtrnoicsnatpir

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.

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2 

Zusammenfassung

Zusammenfassung

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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 desIFNγPromotors, 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.

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3 

Introduction

Introduction

3.1 Kaposis sarcoma-associated herpesvirus

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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 cancer 1 . 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 epidemic 2 . 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 cells 3 and the multicentric Castleman disease 4 . The prevalence of  KSHV is below 10% in western countries 5 , but raises up to 90% in sub-Saharan Africa 6;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 progeny 9 .

KSHV belongs to theγ 2 -herpesvirus subgroup. Its closest relatives are the monkey viruses herpesvirus saimiri (HVS) and rhesus rhadinovirus (RRV); the nearest human pathogenic relative is theγ 1 -herpesvirus Epstein Bar virus (EBV) 10 . The double stranded DNA genome  of KSHV consist of around 165 kb and contains more than 80 open reading frames (ORFs) 11 ORFs 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 evolution 12 . Among them is a cluster of four  genes with

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7

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Introduction

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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 differentiation 13 . 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 / G NGAAANNGAAACT) in responsive promoters 14 . The less conserved IAD mediates interaction with other IRFs for the formation of homo- and heterodimers or interacts with other transcription factors 15 .

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

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Introduction

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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 exons 19 , (2) two alternative splice sites in the sixth exon 20  and (3) two different poly-A sites 21 . 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 IRF5 22 . KSHV positive PEL cell lines express the IRF5 isoforms V1, V2, V3/4 and V5 23 .

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 described 24 . IRF5 possesses a constitutive nuclear localization signal (NLS) and a stronger nuclear export signal (NES), so that IRF5 levels in the nucleus are normally low 25 ; 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 410/411 27 . This ubiquitinylation is required for nuclear translocation 27 . Furthermore, IRF5 is phosphorylated by IKKα 28 , IKKε TBK-1 and 25 ; phosphorylation by IKKα inhibits

TRAF6 mediated ubiquitinylation, whereas the phosphorylation by IKKεand TBK-1 seems to be necessary for dimerization 29 . Phosphorylated nuclear IRF5 can bind to

DNA as homodimer or as heterodimer with IRF3 or IRF7. Thereby, activation of

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