The rabies virus phosphoprotein P: a key regulator of innate immune responses Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München zur Erlangung des Dr. rer. nat. vorgelegt von Krzysztof Brzózka München, April 2006 Erstgutachter: PD Dr. Bettina Kempkes Zweitgutachter: Prof. Dr. Michael Boshart Sondergutachter: Prof. Dr. Karl-Klaus Conzelmann Tag der mündlichen Prüfung: 22 November 2006 TABLE OF CONTENTS 1 TABLE OF CONTENTS 1 TABLE OF CONTENTS ....................................................................................................................... 3 2 ABBREVIATIONS ................................................................................................................................4 3 INTRODUCTION .................................................................................................................................. 6 3.1 RABIES VIRUS (RV) ........................................................................................................................ 6 3.1.1 Pathogenicity ......................................................................................................................... 6 3.1.2 Virus structure and replication............................................................................................. 7 3.1.3 Multifunctional phosphoprotein P ....................................................................................... 9 3.2 INNATE IMMUNITY: INTERFERON .................................................................................................... 11 3.2.1 Interferon induction............................................................................................................. 11 3.2.1.1 Interferon regulatory factors...................................................................................... 13 3.2.1.2 RNA-helicase pathway................................................................................................ 14 3.2.1.3 Toll-like receptor 3 pathway....................................................................................... 15 3.2.1.4 Toll-like receptor 7/9 pathway.................................................................................... 16 3.2.2 Interferon signaling ............................................................................................................. 17 3.2.3 Interferon stimulated genes (ISGs).................................................................................... 19 3.3 VIRAL INHIBITORS OF INNATE IMMUNE RESPOSE ............................................................................. 20 3.3.1 Viral inhibitors of IFN induction ......................................................................................... 20 3.3.2 Viral inhibitors of interferon signaling .............................................................................. 21 4 DISCUSSION...................................................................................................................................... 23 4.1 INHIBITION OF INTERFERON BETA INDUCTION BY RABIES VIRUS P PROTEIN...................................... 23 4.2 INHIBITION OF INTERFERON SIGNALING BY RABIES VIRUS P PROTEIN............................................... 27 4.3 CONCLUDING REMARKS................................................................................................................ 32 5 SUMMARY ......................................................................................................................................... 34 6 ZUSAMMENFASSUNG ..................................................................................................................... 36 7 REFERENCE LIST............................................................................................................................. 38 8 APPENDIX: ........................................................................................................................................ 48 8.1 ARTICLES ENCLOSED AS A PART OF THE CUMULATIVE DOCTORAL THESIS: ...................................... 48 8.2 ACKNOWLEDGEMENTS ................................................................................................................. 49 8.3 CURRICULUM VITAE ..................................................................................................................... 50 ABBREVIATIONS 2 ABBREVIATIONS AP1 activator protein 1 Akt alpha serine/threonine-protein kinase BDV Borna disease virus CARD caspase activation and recruitment domain Cardif CARD adapter inducing interferon-beta CBP cAMP-responsive-element-binding protein (CREB)-binding protein CNS central nervous system CpG cytidine-phosphate-guanosine CRM1 chromosome region maintenance 1 dsRNA double stranded RNA dsDNA double stranded DNA GAF gamma-activated factor GAS gamma-activated sequence IFN interferon IFNAR interferon alpha receptor IFNGR interferon gamma receptor IKK IκB kinase IL interleukin IPS-1 interferon-beta promoter stimulator 1 IRAK IL-1 receptor associated kinase ISG interferon stimulated gene ISGF3 IFN-stimulated gene factor 3 ISRE interferon stimulated response element IRF interferon regulatory factor JAK Janus kinase Lgp2 probable ATP-dependent helicase MAVS mitochondrial antiviral signaling protein MDA5 melanoma differentiation-associated gene 5 MHC major histocompatibility complex MyD88 myeloid differentiation primary response protein 88 NFκB nuclear factor kappa-B NNSV non-segmented negative strand RNA viruses 2’-5’OAS 2’-5’oligoadenylate synthetase pDC plasmacytoid dendritic cell PAMP pathogen-associated molecular patterns PI3K phosphoinositide-3 kinase PKR protein kinase R PML promyelocytic leukemia protein RIG-I retinoic acid inducible gene-I RNP ribonucleoprotein RSV respiratory syncytial virus RV rabies virus ssRNA single strand RNA SOCS suppressor of cytokine signaling STAT signal transducer and activator of transcription TANK TRAF-associated NFκB kinase TBK-1 TANK-binding kinase 1 TIR Toll/IL-1 receptor TNF tumour necrosis factor TRAF TNF Receptor-Associated Factor TRIF Toll/IL-1R domain-containing adaptor inducing IFN-β ABBREVIATIONS TRAM TRIF-related adaptor molecule TLR Toll-like receptor VISA virus-induced signaling adaptor VSV vesicular stomatitis virus VV vaccinia virus 5 INTRODUCTION 3 INTRODUCTION 3.1 Rabies virus (RV) 3.1.1 Pathogenicity Rabies belongs to one of the oldest known infectious diseases. Already first known reports that are found in Egyptian writings, associate the consequences of a contact with a “mad” dog with an acute, progressive and incurable encephalitis (Hemachudha et al., 2002; Rupprecht et al., 2002). Rabies is caused by a neurotropic RNA virus of the Rhabdoviridae family, genus Lyssavirus. Bites and scratches represent the typical transmission route of this virus. After initial replication in the peripheral wound RV is transported in a retrograde way to the central nervous system (CNS). The incubation period ranges from one week to several months. The neurotropism of RV is at least in part caused by the use of several receptors in the CNS to facilitate virus entry into neurons: the neural cell adhesion molecule (Thoulouze et al., 1998), the p75 neurotrophin receptor (p75NTR) (Tuffereau et al., 2001; Tuffereau et al., 1998) and the acetylcholine receptor (Lentz et al., 1982). Rabies is characterized by very little neuronal pathology and mild CNS inflammation. Clinical presentation of rabies disease comes in two major forms, encephalitic (furious) and paralytic (dumb), but there is no clear explanation for this dysfunction of the limbic system. Direct post exposure treatment encompasses wound treatment, vaccine administration and inoculation of rabies virus neutralizing immunoglobulins. This treatment is mostly effective when applied in time, but once first symptoms of rabies encephalitis occur, the outcome of the disease is almost always fatal and the management of the disease is palliative (Jackson et al., 2003; Warrell and Warrell, 2004). Recent studies addressed the question of rabies virus pathogenicity and activation of the immune response system. Inflammation and production of neutralizing antibodies, which depend on B lymphocytes and CD4+ T cells, are crucial for clearance of RV from the CNS (Dietzschold, 1993; Dietzschold et al., 1992; Perry and Lodmell, 6 INTRODUCTION 1991; Hooper et al., 1998). As mature neurons are relatively resistant to either cell or cytokine induced cytolysis, the role of cytotoxic CD8+ T cells in RV clearance is considered minor. A major role can be attributed to cytokine production, mainly IFN-γ. Infection with pathogenic rabies virus results in chemokine production and infiltration of the CNS by mononuclear inflammatory cells (NK cells, T and B lymphocytes). In contrast to attenuated RV, no or only slight activation of innate immune response was observed (Wang et al., 2005; Nakamichi et al., 2004; Nakamichi et al., 2005; Lafon, 2005). Additionally, rabies virus infected neurons retain their integrity but upregulate FasL levels, and thereby induce apoptosis of T-cells, shortly after the T-cells cross the blood- brain barrier (Baloul et al., 2004). 3.1.2 Virus structure and replication RV is a prototypic virus of the Mononegavirales order, the nonsegmented negative strand RNA viruses (NNSV). The RV single strand RNA genome of approximately 12 kb is packed into a bullet-shaped, enveloped virion of approximately 250 nm length and 70 nm width (Figure 1). It comprises five genes encoding nucleoprotein (N), phosphoprotein (P), matrixprotein (M), glycoprotein