Viral modulation of host cell anti-apoptotic mechanisms and cellular stress to promote viral pathogenesis Johanna Viiliäinen Department of Biosciences Genetics Faculty of Biological and Environmental Sciences University of Helsinki ACADEMIC DISSERTATION To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences of the University of Helsinki in the lecture hall 2 at Biomedicum helsinki, (Haartmaninkatu 8, 00290 Helsinki), on September 23rd 2016 at 12 o´clock HELSINKI 2016 Supervisors: Professor Päivi Ojala Giuseppe Balistreri, PhD Research Programs Unit Translational Cancer Biology Faculty of Medicine University of Helsinki Thesis committee members: Professor emeritus Olli Jänne Docent Carina Holmberg-Still Institute of Biomedicine Faculty of Medicine University of Helsinki Pre-examiners: Docent Sisko Tauriainen Department of virology University of Turku Professor Jukka Westermarck Turku Centre for Biotechnology University of Turku Opponent: Docent Varpu Marjomäki Division of Cell and Molecular Biology Department of Biological and Environmental Science University of Jyväskylä Custos: Professor Minna Nyström Division of Genetics Department of Biosciences University of Helsinki ISBN 978-951-51-2397-8 (paperback) ISBN 978-951-51-2398-5 (PDF) http://ethesis.helsinki.fi Helsinki 2016 “YOUR FOCUS DETERMINES YOUR REALITY .”-QUI-GON JINN CONTENTS CONTENTS ORIGINAL PUBLICATIONS ......................................................................................................... i ABBREVIATIONS ....................................................................................................................... iii ABSTRACT .................................................................................................................................. vi REVIEW OF THE LITERATURE ................................................................................................. 1 1. Programmed cell death ............................................................................................................ 1 1.1. Caspases ........................................................................................................................ 1 1.2. Intrinsic pathway ............................................................................................................... 2 1.3. Extrinsic pathway .............................................................................................................. 2 1.4. Anti-apoptotic mechanisms ............................................................................................... 3 1.5. Signaling pathways regulating apoptosis ........................................................................... 4 1.6. Apoptosis in cancer ........................................................................................................... 5 2. Cell cycle................................................................................................................................. 7 2.1. Cdk inhibitory proteins ...................................................................................................... 9 3. DNA damage response (DDR) ............................................................................................... 11 4. Tumor suppressor p53 ........................................................................................................... 12 4.1. The role of p53 in apoptosis ............................................................................................ 12 4.2. The role of p53 in cell cycle arrest ................................................................................... 13 4.3. Modulation of p53 activity .............................................................................................. 13 4.4. Regulation of p53 by Mdm2 ............................................................................................ 14 4.5. Regulation of Mdm2 ....................................................................................................... 15 5. Kaposi’s sarcoma herpesvirus (KSHV) .................................................................................. 16 5.1. Discovery of KSHV and its associated malignancies ....................................................... 16 5.1.1. Kaposi’s sarcoma ......................................................................................................... 16 5.1.2. Primary effusion lymphoma ......................................................................................... 17 5.1.3. Other KSHV associated diseases .................................................................................. 18 5.2. KSHV biology ................................................................................................................ 19 5.2.1. Taxonomy .................................................................................................................... 19 5.2.2. Viral transmission and infection rates ........................................................................... 19 5.2.3. Virion structure and viral entry ..................................................................................... 19 5.2.4. KSHV life cycle ........................................................................................................... 21 5.2.5. Establishment of latent infection................................................................................... 21 5.2.6. Reactivation from latency and the lytic cycle ................................................................ 22 5.2.7. Regulation of reactivation............................................................................................. 25 5.2.7.1. Chromatin modifications controlling the KSHV life cycle ......................................... 25 CONTENTS 5.2.7.2. Chemical induction .................................................................................................... 27 5.2.7.2.1. Phorbol ester TPA .................................................................................................. 27 5.2.7.2.2. HDAC inhibitors .................................................................................................... 27 5.2.7.3. Natural inducers of reactivation ................................................................................. 28 5.2.7.3.1. Hypoxia .................................................................................................................. 28 5.2.7.3.2. Oxidative stress ...................................................................................................... 29 5.3. Modulation of apoptosis and cell cycle in latency ............................................................ 29 5.3.1. Manipulation of cell cycle during the lytic phase .......................................................... 30 5.3.2. Manipulation of apoptosis during the lytic phase .......................................................... 31 5.4. The lytic replication phase is necessary for the progression of KSHV associated malignancies .......................................................................................................................... 32 5.5. Latest discoveries on therapeutic modalities of KSHV associated cancers ....................... 33 AIMS OF THE STUDY................................................................................................................ 36 MATERIALS AND METHODS................................................................................................... 37 RESULTS AND DISCUSSION .................................................................................................... 47 1. KSHV-encoded miRNAs target effector caspase 3 to inhibit apoptosis in latently infected cells (I) .............................................................................................................................................. 47 1.1 KSHV-encoded miRNAs target caspase 3............................................................................ 47 1.2 Depletion of KSHV miRNAs upregulates caspase 3 expression in KSHV-infected PEL cells .................................................................................................................................................. 48 1.3 KSHV miRNAs inhibit apoptosis in KSHV-infected endothelial cells ................................. 49 2. Targeting cellular anti-apoptotic factors to specifically kill virus-infected cells (II, unpublished data) .......................................................................................................................................... 50 2.1 KSHV-infected cells are resistant to the Bcl-2 inhibitor ABT-263........................................ 50 2.2 ABT-263 did not induce cell death of the lytic KSHV-infected cells .................................... 52 2.3 Obatoclax induces cell death in the lytic KSHV-infected cells ............................................. 54 3. KSHV utilizes the p53 stress response to induce a G2/M cell cycle checkpoint for efficient lytic replication (III) .................................................................................................................. 56 3.1 siRNA screen identifies MDM2 as a novel regulator of KSHV reactivation ......................... 56 3.2 Knockdown of MDM2 enhances KSHV reactivation ........................................................... 56 3.3 Viral reactivation leads to a p53 response in PEL cells ......................................................... 57 3.4 p53 is required at the early phase of the reactivation for efficient lytic replication ................ 58 3.5 p53 is required to induce p21Cip-1-mediated G2/M cell cycle arrest upon KSHV reactivation 59 4. Characterization of the protein-protein interactome of v-cyclin and host cell proteins ............ 62 4.1 Generation of tetracycline inducible isogenic v-cyclin expressing human cell line ............... 62 4.2 Affinity purification of v-cyclin and its binding proteins ...................................................... 63 CONTENTS 4.3 Identification of the v-cyclin interacting proteins by AP LC-MS/MS ................................... 65 4.4 Validation of novel v-cyclin interacting proteins by Western Blot ........................................ 67 CONCLUSIONS AND FUTURE PROSPECTS ........................................................................... 70 ACKNOWLEDGEMENTS .......................................................................................................... 72 BIBLIOGRAPHY ......................................................................................................................... 74 ORIGINAL PUBLICATIONS ORIGINAL PUBLICATIONS This thesis is based on the following original publications, which are referred to in the text by their Roman numerals. I. Suffert G*, Malterer G*, Hausser J‡, Viiliäinen J‡ , Fender A, Contrant M, Ivacevic T, Benes V, Gros F, Voinnet O, Zavolan M, Ojala PM**, Haas JG**, Pfeffer S**.: Kaposi's sarcoma herpesvirus microRNAs target caspase 3 and regulate apoptosis. PLoS Pathog. 2011 Dec;7(12):e1002405. * These authors contributed equally to the work and should be considered as joint first authors ‡ These authors contributed equally to the work and should be considered as joint second authors ** shared correspondence II. Kakkola L*, Denisova OV*, Tynell J, Viiliäinen J, Ysenbaert T, Matos RC, Nagaraj A, Ohman T, Kuivanen S, Paavilainen H, Feng L, Yadav B, Julkunen I, Vapalahti O, Hukkanen V, Stenman J, Aittokallio T, Verschuren EW, Ojala PM, Nyman T, Saelens X, Dzeyk K, Kainov DE.: Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice. Cell Death Dis. 2013 Jul25;4:e742. * These authors contributed equally to the work and should be considered as joint first authors III. Balistreri G*, Viiliäinen J*, Turunen M ‡, Diaz R ‡, Lyly L, Pekkonen P, Rantala J, Ojala K, Sarek G, Teesalu M, Denisova O, Peltonen K, Julkunen I, Varjosalo M, Kainov D, Kallioniemi O, Laiho M, Taipale J, Hautaniemi S, Ojala PM.: Oncogenic Herpesvirus utilizes stress-induced cell cycle checkpoints for efficient lytic replication. PLoS Pathog. 2016 Feb 18;12(2):e1005424. * equal contribution ‡ equal contribution Additional unpublished material is also presented. i ORIGINAL PUBLICATIONS Contributions to the original publications I: Generation and use of KSHV miRNA expressing cell models representing biologically relevant model systems for KSHV infection and to be used in functional assays for addressing the role of miRNA expression in apoptosis inhibition (Figure 7D-G and 8). Contributed to preparation of the figures and writing the manuscript. II: Experiments involving KSHV-infected cells. Contributed to preparation of the figures and writing the manuscript. III: Preparation of the rKSHV-SLK cell line and siRNA screen siRNA screen together with Juha Rantala at VTT, Turku siRNA transfection of iSLK.219 cells and measurement of RFP positive cells by using an automated high-content fluorescence microscope (CellInsight, Thermo Scientific) Lentivirus production and transduction validations of five different shMDM2 expressing lentiviruses. Preparation of sh-Scr and two independent sh-MDM2 expressing BC-3 cells by lentiviral transduction, lytic replication induction by TPA treatment followed by qRT-PCR analyses. Preparation of samples for ChIP-Seq by TPA and Nutlin induction of BC-3 cells and immunoblotting of input samples confirmation for p53 stabilization and induction of p21 expression. Maintenance and TPA induction of stable sh-Scr and sh-p53 BC-3 cells followed by immunoblotting and qRT-PCR analysis to address p53 dependency of viral reactivation. Contributed to design of the experiments and preparation of the figures. ii ABBREVIATIONS ABBREVIATIONS 3´UTR AP Apaf-1 APC/C ATM ATR Bad Bak Bap1 Bax BCBL Bcl-2 BCLAF1 Bcl-w Bcl-xL Bim BRCAI CAK cART CBP Cdc25 Cdk CDKN1A cFLIP cFLIPL cFLIPS ChIP-seq Chk2 CKI CMV c-Myc CREB CSMA CTG DBD DD DDR DE DED DISC DNA-PK DNMT DSB E2F EBV ECM EGFR ERK 3´Untranslated region affinity purification apoptotic protease-activating factor 1 anaphase promoting complex ataxia telangiectasia mutated ataxia telangiectasia and Rad3related protein Bcl-2 antagonist of cell death Bcl-2 antagonist killer BRCA1 associated protein-1 Bcl-2-associated X protein body cavity-based lymphoma B-cell lymphoma 2 Bcl-2-associated factor Bcl-2-like protein 2 B-cell lymphoma-extra large Bcl-2-interacting mediator of cell death breast cancer susceptibility gene 1 Cdk-activating kinase combination antiretroviral treatment CREB binding protein Cell division cycle 25 cyclin dependent kinase cyclin dependent kinase inhibitor 1A cellular FLICE-inhibitory protein long form of FLIP short form of FLIP Chromatin immunoprecipitation sequencing checkpoint kinase 2 Cdk inhibitory protein cytomegalovirus V-Myc Avian Myelocytomatosis Viral Oncogene Homolog cAMP response element-binding protein cell-spot microarray Cell titer glo assay DNA binding domain death domain FADD FoxO3a G1 G2 GFP GO H2O2 HA HAART HAT HDAC HGF HHV HIF HIV-1 HMT hpi HR HRE HUVEC IAP IE IF IKZF1/3 IL-2 IL-3 iLEC IMiD IR iii DNA damage response delayed-early gene death effector domains death-inducing signaling complex DNA-dependent protein kinase DNA-methyl transferase double stranded break E2 factor Epstein-Barr virus extra cellular matrix epidermal growth factor receptor Extracellular signal-regulated kinase Fas-associated DD protein forkhead box O3a Gap 1 Gap 2 G2 green fluorescent protein Gene Ontology hydrogen peroxide Hemagglutinin highly active antiretroviral therapy histone acetyltransferase histone deacetylase Hepatocyte growth factor human herpesviruses hypoxia-inducible factor human immunodeficiency virus histone methyl transferases hours post induction homologous recombination hypoxia response element Human umbilical vein endothelial cells Inhibitor of Apoptosis proteins immediate early gene indirect immunofluorescence Ikaros family zinc finger 1 and 3 Interlukin-2 interleukin-3 immortal lymphatic endothelial cell Immunomodulatory drug ionizing radiation ABBREVIATIONS iSLK.219 IκB JMJD3 JNK KAP1 KICKS K-iLEC KS KSHV L LANA MCD Mcl-1 MDM2 miRNA MLL2 MM M-phase MTA mTOR Myt1 NaB NAC NAM NES NF-κB NHEJ NHL NLS Noxa PAN PBMC PCNA PEL pH3 S10 PI3K PIKK PKA PKC pRb PRC2 PUMA rKSHV.219-infected SLK cell clone inhibitor κB Jumonji domain containing-3 C-Jun N-terminal protein kinase KRAB-associated protein-1 KSHV-associated inflammatory cytokine syndrome KSHV-infected lymphatic endothelial cell line Kaposi’s sarcoma Kaposi’s sarcoma herpesvirus late gene Latency Associated Nuclear Antigen multicentric Castleman’s disease myeloid cell leukemia-1 Mouse double minute-2 microRNAs histone-lysine N-methyltransferase 2 primary rat mesenchymal precursor cell line mitosis mRNA transport and accumulation protein mammalian target of rapamycin Myelin transcription factor 1 sodium butyrate N-acetyl cysteine nicotinamide nuclear export signal nuclear factor kappa-B non-homologous end joining non-Hodgkin’s lymphoma nuclear localization sequence PMA-Induced Protein polyadenylated nuclear RNA peripheral blood mononuclear cell proliferating cell nuclear antigen primary effusion lymphoma histone 3 phosphorylated on serine 10 Phosphoinositide 3-kinase phosphatidyl inositol 3 kinase-like protein kinase protein kinase A protein kinase C RAD51AP1 RAP RBP-Jκ RFP rKSHV.219 ROS RRE RT RTA S6KB1 SAC shRNA Sp1 S-phase SPP1 SSB SSBR ssDNA SV40 SWI/SNF TAD TAP tBid TET TF TGFβ tinyLNA TNFR TNFα TPA TR TRADD TRAIL TSA TUNEL UTX iv retinoblastoma protein Polycomb repressive complex 2 p53 upregulated modulator of apoptosis RAD51-associated protein replication-associated protein Recombining binding protein suppressor of hairless red fluorescent protein recombinant KSHV reactive oxygen species RTA responsive elements room temperature Replication and transcription activator S6 kinase spindle assembly checkpoint short hairpin RNA specifity protein 1 synthesis phase osteopontin single stranded break single-strand break repair single stranded DNA Simian virus 40 SWItch/Sucrose Non-Fermentable transcriptional activation domain tandem affinity purification truncated Bid tetramerization domain transcription factor transforming growth factor β Locked Nucleic Acid oligonucleotide tumor necrosis factor receptor tumor necrosis factor α 12-O-tetradecanoylphorbol-13acetate terminal repeat TNFR-associated DD protein TNF-related apoptosis-inducing ligand trichostatin A TdT-mediated dUTP nick end labeling Ubiquitously transcribed tetratricopeptide repeat gene on X chromosome ABBREVIATIONS UV vBcl2 v-cyclin VEGF vFLIP vGPCR vIL-6 vIRF VPA vPK VZV XBP-1 XIAP ultraviolet radiation viral Bcl-2 viral cyclin vascular growth factor viral FLIP viral protein-coupled receptor viral interleukin-6 viral interferon regulator factor valproic acid viral protein kinase varicella zoster virus X-box binding protein X-linked inhibitor of apoptosis v ABSTRACT ABSTRACT Kaposi’s sarcoma herpesvirus (KSHV) is a human gamma2-herpesvirus and the causative agent of three human cancers: Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV establishes persistent life-long infection and manipulates host cell proliferation and apoptosis, which leads to the development of cancer in immunocompromised individuals. KSHV has two phases of life cycle, the latency phase and lytic replication phase. Latency is a silent mode of infection, while in the lytic replication phase new virus progeny are produced. Although the latent phase is the default mode of infection, the lytic cycle has an instrumental role in the emergence and progression of KS. KSHV encodes several microRNAs (miRNAs) that have a potential role in viral tumorigenesis. However, the targets of these miRNAs are still poorly known. In the first study we show that KSHV-encoded miRNAs inhibit apoptosis by silencing the expression of caspase 3, an apoptotic effector, in latent KSHV-infected cells. In the second study the aim is to discover new specific anti-viral drugs by targeting host cell antiapoptotic mechanisms utilized by KHSV. To this end, BH3 mimetic drugs are tested which bind and inhibit the anti-apoptotic function of the B-cell lymphoma 2 (Bcl-2) family of proteins in KSHV-infected cells. This study shows that the myeloid cell leukemia-1 (Mcl-1) inhibitor, Obatoclax, induces cell death in KSHV-infected cells undergoing lytic phase. The cellular mechanisms that regulate the triggering of the lytic cycle (reactivation) are not completely understood. To discover new regulators of KSHV reactivation and lytic cycle activation, a targeted RNAi screen was performed using a library of siRNA oligos targeting human epigenetic factors and their regulators. The screen identifies Mouse double minute-2 (MDM2), the main regulator of p53 protein stability, as a negative regulator of KSHV reactivation. Further studies demonstrate that efficient KSHV reactivation is favored by a p53 stress response and by the induction of p21Cip-1-mediated G2/M cell cycle arrest. KSHV encoded cyclin D homolog viral cyclin (v-cyclin) is able to overcome G1 cell cycle arrest and dysregulate mitosis of KSHV infected cells. V-cyclin operates by interacting with cellular proteins and consequently to study the role of v-cyclin in KSHV induced cancers, comprehensive interactome of v-cyclin and cellular proteins was studied in tetracycline inducible isogenic v-cyclin expressing human cell line. vi ABSTRACT In summary, research in this thesis identifies a new cellular target of KSHV-encoded miRNAs and uncovers a cellular anti-apoptotic mechanism utilized by this virus to protect cells undergoing lytic replication from premature death. Inhibition of this mechanism enabled the identification of a potential new drug candidate against KSHV-associated cancers. Additionally, this study provides new insights into how KSHV can cope with and utilize cellular stress response to ensure efficient lytic replication and revealed 8 novel v-cyclin interacting cellular proteins. vii REVIEW OF LITERATURE REVIEW OF THE LITERATURE Like other herpesviruses, KSHV infection results in extensive alterations of cellular processes such as apoptosis, the DNA-damage response and cell cycle progression (Mesri, Cesarman et al. 2010). The molecular details of these alterations, the implications for virus replication and associated diseases, and the possibility to interfere with these processes as a potential therapeutic strategy are the main topics of this thesis. 1. Programmed cell death Apoptosis is a tightly regulated cellular process leading to cell death. It is characterized by a series of changes in cell morphology and structure, including cell membrane blebbing, the collapse of the nucleus into dense structures, and the fragmentation of the chromosomal DNA (Kerr, Wyllie et al. 1972, Kroemer, El-Deiry et al. 2005) and of the whole cell to form apoptotic bodies. These apoptotic bodies are 1-5 m vesicular structures that are rapidly ingested by neighboring cells or by macrophages due to the exposure of the signaling lipid phosphatidylserine (Golstein 1998, Hengartner 2000). Apoptosis is a physiological process that occurs during embryogenensis, lymphocyte development and tissue maintenance, and can also be induced by several cell stresses such as oncogene hyperactivation, virus infection, Ultraviolet (UV) radiation and DNA damage. The main executors of the apoptotic program are cysteine proteases called caspases (Yuan, Shaham et al. 1993, Nicholson, Ali et al. 1995). These enzymes can be activated by the intrinsic (mitochondrial-mediated) or extrinsic (death receptor-mediated) apoptotic pathways (Figure 1). 1.1. Caspases Caspases are synthesized in an inactive zymogenic pro-enzyme form and are activated by proteolytic cleavage. Caspases consist of two domains: the N-terminal pro-domain, which is important for caspase regulation, and the C-terminal protease domain, which carries catalytic activity. Apoptotic caspases can be divided into two groups: initiators and effectors. Initiator caspases, such as caspases-8 and -9, are activated by adaptor proteins which interact with the prodomain and promote caspase dimerization and activation (Boatright, Renatus et al. 2003, Pop, Timmer et al. 2006). Effector caspases, such caspases-3and -6, have a shorter pro-domain and are activated by the initiator caspases through proteolytic cleavage. Once activated, effector caspases cleave cellular substrates such as lamin and Poly (ADP-ribose) polymerase (PARP). These and other cleavage events initiate the dismantling of the cell (Mahrus, Trinidad et al. 2008). 1 REVIEW OF LITERATURE 1.2. Intrinsic pathway The intrinsic apoptosis pathway (Figure 1) refers to programmed cell death initiated by a series of reactions that lead to the disruption of the mitochondrial membrane and the release of cytochromeC into the cytoplasm, which induces the activation cascade of caspases starting from initiator caspases such as caspases-2, -8, -9 and -10 (Ashkenazi and Salvesen 2014) . Intrinsic apoptosis is regulated by the Bcl-2 family of proteins, which includes both pro- and antiapoptotic members. Whether they induce or inhibit apoptosis depends on what structural domains they contain. The type and number of structural domains are used to categorize Bcl-2 family members into three functional sub-groups: i) initiators of apoptosis or "BH-3-only" proteins ii) proapoptotic effector proteins and iii) anti-apoptotic proteins. Apoptotic signals such as DNA damage and growth factor deprivation activate the initiator BH3-only proteins such as Bcl-2-interacting mediator of cell death (Bim) and Bcl-2-antagonist of cell death (Bad), which inactivate antiapoptotic Bcl-2 family proteins such as Bcl-2, B-cell lymphoma-extra large (Bcl-X L) and Mcl-1. This results in the activation of the pro-apoptotic effector proteins Bcl-2-associated X protein (Bax) and Bcl-2 antagonist killer (Bak). Active Bax and Bak are proteins that form pores on the outer mitochondrial membrane causing the release of cytochrome C (Czabotar, Lessene et al. 2014). Released cytochrome C induces the formation of the apoptosome, a large protein complex that includes apoptotic protease-activating factor 1 (Apaf-1) and is responsible for recruiting and activating procaspase-9 (Li, Nijhawan et al. 1997, Kroemer, Galluzzi et al. 2007). Activated caspase-9 cleaves and activates effector caspase-3, which cleaves other effector caspases, including -6 and -7, leading to the execution of apoptosis. 1.3. Extrinsic pathway Extrinsic apoptosis (Figure 1) is induced by the activation of plasma membrane receptors called death receptors. These receptors belong to the tumor necrosis factor receptor (TNFR) superfamily and contain the death domain (DD). The death receptors which can induce apoptosis via caspase activation are TNF Receptor Superfamily, Member 6 (Fas), TNFR1, death receptor 3 (DR3), death receptor 4 (DR4), death receptor 5 (DR5) and death receptor 6 (DR6) (Locksley, Killeen et al. 2001, Lavrik, Golks et al. 2005). Binding of death ligands such as Fas (FasL) and tumor necrosis factor α (TNFα) causes a conformational change of the death receptor and exposes the DD domain, which recruits adaptor proteins such as Fas-associated DD protein (FADD) and TNFR-associated DD protein (TRADD). Adaptor proteins bind pro-caspase-8 (also known as FLICE) through their death effector domains (DED) and form the death-inducing signaling complex (DISC), which triggers 2 REVIEW OF LITERATURE caspase activation (Wallach, Varfolomeev et al. 1999, Schneider and Tschopp 2000, Wong 2011). Activated caspase-8 cleaves and activates effector caspases 3, 6 and 7 (Hirata, Takahashi et al. 1998, Kruidering and Evan 2000). Additionally, caspase-8 can cleave Bid, forming truncated Bid (tBid), which translocates to the mitochondria and causes release of cytochrome C into the cytoplasm (Li, Zhu et al. 1998, Luo, Budihardjo et al. 1998). Figure 1. Intrinsic and extrinsic apoptosis pathways. 1.4. Anti-apoptotic mechanisms The extracellular environment and the signals the cell receives from it determine whether it dies or survives. Biochemically, it is defined by the balance of pro-apoptotic and anti-apoptotic factors. Extracellular signals such as growth factors, chemokines, hormones and contacts with the extra cellular matrix (ECM) typically promote cell survival by activating intracellular anti-apoptotic factors. These anti-apoptotic factors function at multiple levels, inhibiting both extrinsic and intrinsic apoptosis pathways by antagonizing caspases or pro-apoptotic Bcl-2 proteins, or by 3 REVIEW OF LITERATURE promoting the expression of anti-apoptotic genes after having activated the transcription factor nuclear factor kappa-B (NF-κB) (Portt, Norman et al. 2011). Apoptosis can be inhibited by Inhibitor of Apoptosis proteins (IAP), such as X-linked inhibitor of apoptosis (XIAP), which can bind and inhibit caspases-3, -7 and -9. Pro-apoptotic stimuli cause the release of second mitochondria-derived activator of caspases (SMAC/DIABLO) from the mitochondria, which inhibits the IAPs to promote cytochrome C-induced caspase activation (Deveraux and Reed 1999, LaCasse, Mahoney et al. 2008). Cellular FLICE-inhibitory protein (cFLIP) is another inhibitor of apoptosis and is homologous to procaspase-8. cFLIP contains two DED-domains, which enable it to bind to the FADD and the DISC complex. cFLIP has two mRNA isoforms: the short form (cFLIP S) and the long form (cFLIPL). cFLIPS lacks a catalytic domain, thus it competes with procaspase-8 for DISC binding and hence inhibits procaspase-8 cleavage. The cFLIP L variant can heterodimerize with procaspase-8 and promote its activation (Chang, Xing et al. 2002), however, it has been recently shown that these heterodimers promote cell survival (Oberst, Dillon et al. 2011, van Raam and Salvesen 2012), underscoring the complexity of apoptotic regulation. NF-κB is a transcription factor that regulates cell survival and is activated mainly by TNFα. As mentioned earlier, TNFα induces apoptosis via TNFR, but in addition to that, TNFR can also induce anti-apoptotic pathways by binding to the TNF receptor-associated factor 2 (TRAF2) protein, which in turn recruits Receptor-interacting serine/threonine-protein kinase 1 (RIP1) and activates NF-κB. Additionally, TNFα can activate the Phosphoinositide 3-kinase (PI3K)/Akt/Protein kinase B (PKB) kinase pathway, which results in the phosphorylation and activation of the IκB kinase (IKK) kinase complex, which in turn phosphorylates and inactivates the inhibitor κB (IκB) and causes activation of NF-κB. NF-κB activates the expression of anti-apoptotic genes such as Bcl-X L and XIAP and therefore promotes cell survival (Barkett and Gilmore 1999, Brenner, Blaser et al. 2015). 1.5. Signaling pathways regulating apoptosis Apoptosis and cell survival is regulated by signaling pathways which are responsive to extracellular signals such as mitogens and stress inducers. Extracellular signal-regulated kinase (ERK) and C-Jun N-terminal protein kinase (JNK)/p38 pathways forms regulatory network where ERK promotes cell survival by inhibiting pro-apoptotic function of JNK/p38 pathway whereas JNK/p38 inhibits ERK pathway when encountering stress signals. Stress induce apoptosis via p38/JNK mediated activation of protein phosphatase 2A (PP2A) and MAPK phosphatase (MKP) which negatively regulate ERK 4 REVIEW OF LITERATURE pathway (Junttila, Li et al. 2008). These pathways regulate stability and activity of pro- and antiapoptotic proteins. Phosphorylation status of Bim determinates its function and stability. Phosphorylation of Bim by ERK1,2 lead to its proteasomal degradation whereas phosphorylation by JNK or p38 increase its pro-apoptotic activity (Putcha, Le et al. 2003, Okuno, Saito et al. 2004, Ley, Ewings et al. 2005, Cai, Chang et al. 2006). Furthermore protein kinase A (PKA) activates Bim by phosphorylating cAMP response element-binding protein (CREB) (Zhang and Insel 2004). ERK1,2 mediates cell survival by co-operating with ribosomal S6 kinase (RSK) to phosphorylate Bim which lead to its ubiquitin mediated degradation (Dehan, Bassermann et al. 2009). Furthermore RSK kinase can activate CREB transcription factor which upregulate expression of Bcl-2 and BclxL (Bonni, Brunet et al. 1999). Moreover ERK1,2 is able to phosphorylate and inhibit caspase-9 (Allan, Morrice et al. 2003). ERK signaling is also inhibiting extrinsic apoptosis by inhibiting Fas induced DISC formation (Holmstrom, Schmitz et al. 2000). Also epidermal growth factor receptor (EGFR) activated Src kinase has been found to phosphorylate caspase-8 and prevent extrinsic apoptosis upon growth factor stimulation (Cursi, Rufini et al. 2006). It has been shown that growth promoting cytokine interleukin-3 (IL-3) activates ERK mediated phosphorylation of Bim, which dissociates it from Bax and inhibits its pro-apoptotic function (Harada, Quearry et al. 2004). Also IL-3 activated PI3K/Akt phosphorylates Bad and inhibits apoptosis (Zha, Harada et al. 1996, Datta, Dudek et al. 1997, del Peso, Gonzalez-Garcia et al. 1997). Akt is also downregulating expression of Bim by phosphorylating and inactivating transcription factor (TF) forkhead box O3a (FoxO3a) which is needed for Bim transcription (Dijkers, Medema et al. 2000, Tzivion, Dobson et al. 2011). Growth factor deprivation and cellular stress activates pro-apoptotic proteins. Interlukin-2 (IL-2) deprivation lead to phosphatase type 1 alpha (PP1α) mediated de-phosphorylation and activation of caspase-9 (Dessauge, Cayla et al. 2006) and DNA damage induces phosphorylation of caspase-9 at Tyr153 by c-Abl kinase which leads to activation of caspase-9 and induction of apoptotic response (Raina, Pandey et al. 2005). 1.6. Apoptosis in cancer One of the functions of apoptosis is to eliminate neoplastic and potentially malignant cells (Kerr, Wyllie et al. 1972). Thus, evasion of the apoptotic program is one of the hallmarks of the multistep tumorigenesis process (Hanahan and Weinberg 2000, Hanahan and Weinberg 2011). Cancer cells 5 REVIEW OF LITERATURE can evade apoptosis by multiple mechanisms such as disrupting the balance between anti- and proapoptotic Bcl-2 family proteins, attenuation of caspase function and reduction of death receptor function. In many cancers, anti-apoptotic Bcl-2 family proteins have been found to be overexpressed, which correlates with poor prognosis and resistance to chemotherapy (Tsujimoto, Finger et al. 1984, Raffo, Perlman et al. 1995, Friess, Lu et al. 1998, Taniai, Grambihler et al. 2004, Wuilleme-Toumi, Robillard et al. 2005, Beroukhim, Mermel et al. 2010). Conversely, pro-apoptotic Bcl-2 proteins such as Bax and Bim and p53 upregulated modulator of apoptosis (PUMA) have been found to be downregulated in certain cancers (Rampino, Yamamoto et al. 1997, Beroukhim, Mermel et al. 2010, Ng, Hillmer et al. 2012). Cancer cells can prevent initiation and execution of apoptosis by reducing caspase activity, and it has been reported that caspase levels are reduced in many malignancies. In addition, caspases-8 and -3 are frequently mutated in several cancers (Ghavami, Hashemi et al. 2009). Moreover loss of Apaf-1 expression have been identified in many cancer and it is associated with poor prognosis (Dai, Martinka et al. 2004, Fujimoto, Takeuchi et al. 2004, Wang, Bai et al. 2007, Zlobec, Minoo et al. 2007) Cancer cells can evade death signals by downregulating the expression of death receptors or impairing their function, and additionally, they can decrease the secretion of death ligands (Debatin and Krammer 2004). Conventional cancer therapies such as chemotherapy and γ-irradiation aim at inducing apoptosis in cancer cells, but defective apoptotic mechanisms of tumor cells may cause drug resistance. Therefore targeting apoptotic mechanisms could provide new therapeutic modalities to fight cancer and currently one of the most promising sets of targets seems to be the Bcl-2 family of proteins. Small-molecule inhibitors such as ABT-737, ABT-263, ABT-199 and Obatoclax are BH-3 mimetic drugs, which induce apoptosis by binding Bcl-2, Bcl-X L, Bcl-w and Mcl-1, preventing their interaction with proapoptotic proteins (Wong 2011, Moldoveanu, Follis et al. 2014). The apoptotic function of ABT737 is synergized with kinase inhibitors as in the study where ABT-737 was combined with kinase inhibitor Sorafinib, co-treatment suppressed growth of hepatocellular cancer cells and xenograft tumors (Hikita, Takehara et al. 2010). And furthermore inhibition of PI3K/ mammalian target of rapamycin (mTOR) pathway synergize ABT-737 apoptotic effect on ovarian cancer cells and myeloid leukemia cells (Rahmani, Aust et al. 2013, Jebahi, Villedieu et al. 2014). ABT-263 is clinical derivative of ABT-737 and it is currently undergoing phase I and II clinical studies in treatment of multiple cancer types (Wilson, O'Connor et al. 2010, Cleary, Lima et al. 2014, Kipps, Eradat et al. 2015). ABT-199 is recently developed derivative, which does not cause thrombocytopenia like ABT-737 and ABT-236 (Souers, Leverson et al. 2013). ABT-199 enhances anti-cancer effect of tamoxifen in breast cancer xenografts (Vaillant, Merino et al. 2013). Moreover ABT-199 was shown to be promising against multiple myeloma and chronic lymphocytic leukemia 6 REVIEW OF LITERATURE (Vogler, Dinsdale et al. 2013, Touzeau, Dousset et al. 2014). Phase I clinical trial to treat relapsed chronic lymphocytic leukemia with ABT-199 revealed manageable safety profile and remarkable response (Roberts, Davids et al. 2016). And importantly ABT-199 is recently approved by food and drug administration (FDA) to treat chronic lymphocytic leukemia patients with chromosome 17p deletion. Obatoclax is inhibiting Bcl-2, Bcl-w, Bcl-xL and Mcl-1 (Nguyen, Marcellus et al. 2007). Obatoclax induce apoptosis, autophagy and necroptosis of acute lymphoblastic leukemia (ALL) cell lines (Urtishak, Edwards et al. 2013) and increased sensitivity of multiple myeloma cells to chemotherapeutic agents (Trudel, Li et al. 2007). 2. Cell cycle Cell division is part of a cellular process referred to as the cell cycle. This highly regulated process consists of four different phases: Gap 1 (G1), synthesis phase (S-phase), Gap 2 (G2) and mitosis (M-phase) (Figure 2). The progression from one phase to the other is mainly coordinated by cyclin proteins and the associated cyclin-dependent kinases (Cdks). The levels of cyclin proteins oscillate during the cell cycle and specific cyclins are expressed in each cell cycle phase (Evans, Rosenthal et al. 1983). Cyclins bind and activate Cdks, which regulate cell cycle progression by phosphorylating their target proteins (Morgan 1995). Cdk activity can be modulated by phosphorylation. Full kinase activity of Cdk-cyclin complex can be reached by phosphorylation by Cdk-activating kinase (CAK) (Nigg 1996). And on the other hand phosphorylation of Cdks at Thr14 and/or Tyr15 residues by Wee1 and Myelin transcription factor 1 (Myt1) kinases inhibit its activity while Cell division cycle 25 (Cdc25) phosphatases can dephosphorylate these residues in order to activate Cdks when required (Malumbres 2014). During G1, a cell grows and prepares itself for the next step of the cell cycle. At the transition between one cell cycle phase and another there exist quality control mechanisms, or checkpoints, to ensure that no 'mistakes' have been made and that cells can proceed towards division. At the G1 checkpoint, cells need to ensure that cell size is sufficient, that the genome is intact, and that an adequate amount of mitogens are present to move into S-phase. Failing this checkpoint activates a restriction mechanism known as the G1 restriction or R-point. G1-phase progression is regulated by cyclin D as well as the Cdk4 and 6 kinases. When sufficient growth factor stimulation exists, cyclin D is expressed and it associates with Cdk4 and 6 (Bottazzi and Assoian 1997). CyclinD/Cdk4 and 6 phosphorylate the retinoblastoma protein (pRb) that gets inactivated (Figure 2). pRb is a 'pocket' protein which acts as a co-repressor together with histone deacetylase 1 (HDAC1) to repress the expression of the transcription factor E2 factor (E2F), which is a main activator of cell cycle progression. Thus, when the repressor pRb is phosphorylated, E2F is de-repressed and is able to initiate transcription of genes required for promoting cell cycle 7 REVIEW OF LITERATURE progression from G1 to S-phase. Some of the targets of E2F include cyclins E and A, as well as proteins required in S-phase for DNA replication (Vermeulen, Van Bockstaele et al. 2003). Cyclin E binds Cdk2 and the active cyclin E/Cdk2 complex is responsible for G1/S progression. During S-phase, the DNA genome of the cell is duplicated and cells need to ensure that this process occurs flawlessly and only once during each cell division. This is monitored during checkpoints that are also in place during S-phase and at the S/G2 transition. After DNA replication is completed, cells enter into G2 phase, where they prepare to enter mitosis by condensing the DNA into chromosomes. If the replicated DNA is intact and the condensed chromosomes formed correctly, cells proceed to mitosis (M-phase). The integrity of replicated DNA is monitored at the onset of the G2 phase, before DNA condensation. If the DNA is damaged, the DNA-damage checkpoint is activated and the cell cycle stops in early G2 to allow DNA repair (Malumbres and Barbacid 2009). Once the damaged DNA is repaired, the accumulated cyclin B binds Ckd1 and cells prepare to enter mitosis. The phosphatase Cdc25 activates the complex by dephosphorylating Cdk1. Mitosis is subdivided into five distinct phases called prophase, prometaphase, metaphase, anaphase and telophase. During prophase, the genome is condensed into chromosomes, and the centrosomes, located at the opposite sides of the nucleus, begin to polymerize microtubules to form the mitotic spindle, a macromolecular structure that segregates the duplicated chromosomes to each side of the dividing cell. During prometaphase, the nuclear envelope is disintegrated, allowing microtubules to attach to the central portion of each chromosome, the kinetochores, forming the mitotic spindle. The mitotic spindle is composed of polar microtubules which are attached from the one end to the centrosome and from the other end to the kinetochore of each chromosome. During metaphase, chromosomes align at the center of the dividing cell, forming the metaphase plate (Malumbres and Barbacid 2009). To ensure correct alignment of chromosomes and secure the proper attachment of kinetochores, mitosis can be stopped by the spindle assembly checkpoint (SAC), which inhibits the anaphase promoting complex (APC/C) protein (Burke and Stukenberg 2008). APC/C is an E3 ubiquitin ligase responsible for the metaphase-to-anaphase transition. APC/C targets cyclins A and B as well as Securin proteins for proteolytic degradation. After securin degradation, separin proteins are activated, enabling segregation of sister chromatids by the hydrolysis of cohesins, which link the sister chromatids together (Peters 2002). Thus, during anaphase, sister chromatids are pulled apart with the help of shortening microtubules. Finally, in telophase, the nuclear envelope reforms around each daughter chromosome and the DNA is decondensed. Thereafter, the two daughter cells separate from each other by a process called cytokinesis. This last step of the cell cycle is achieved 8 REVIEW OF LITERATURE by a contractile ring of actin filaments, which cleaves the cell in two (Malumbres and Barbacid 2009). Figure 2. Regulation of the cell cycle. 2.1. Cdk inhibitory proteins Cdk inhibitory proteins (CKIs) exist in every cell to regulate the cell cycle and prevent abnormal cell proliferation and potential cellular transformation (Figure 2). There are two families of CKIs: INK4 and Cip/Kip. The INK4 family includes the cell cycle progression inhibitors p15 INK4b, p16INK4a, p18INK4c and p19INK4d, which inhibit Cdk4 and 6, causing G1 cell cycle arrest. At the molecular level, the INK4 inhibitors cause a conformational change in Cdk protein structure that inhibits the catalytic activity of the enzyme. Additionally, by binding to the G1-specific Cdks, INK4 inhibitors block the interaction of the kinases with their respective cyclin, cyclin-D (Russo, Tong et al. 1998). Interestingly, INK4 inhibitors are able to arrest the cell cycle in G1-phase only if the E2F inhibitor pRb protein is functional (Bruce, Hurford et al. 2000). Various stress signals in G1 phase can trigger INK4 protein activation. For instance, the expression of p15INK4b and p16INK4a can be activated by oncogenic stress. The transcription factor JunB has been shown to transactivate the expression of p16 INK4a and induce premature senescence of mouse 9 REVIEW OF LITERATURE 3T3 cells (Passegue and Wagner 2000). Moreover, Ets-domain transcription factor (Ets) activates the expression of p16INK4a in senescent human fibroblast cells (Ohtani, Zebedee et al. 2001), whereas p15INK4b expression is induced by transforming growth factor β (TGFβ) in epithelial cells (Hannon and Beach 1994). The Cip/Kip family consists of p21Cip1, p27Kip1 and p57Kip2. Like the INK4 proteins, these inhibitors inactivate Cdk/cyclin complexes, but in a different manner, through binding to both cyclin and Cdk. Also, they seem to have a broader range. They inhibit the G1/S transition by forming a ternary complex with cyclinE/A/Ckd2. In addition, some of the Clip/Kip family members can induce cell cycle arrest in G2 after DNA damage (Bunz, Dutriaux et al. 1998, Smits, Klompmaker et al. 2000). p21Cip1 has also been shown to inhibit DNA replication by inhibiting the activity of proliferating cell nuclear antigen (PCNA) (Li, Waga et al. 1994, Cazzalini, Scovassi et al. 2010). The expression levels of p21Cip1 are mainly regulated by the stress-sensor p53 (a transcription factor) (el-Deiry, Tokino et al. 1993). p27Kip1 expression is regulated by growth inhibitory cytokines, contact inhibition and by several transcription factors like FoxO, specifity protein 1 (Sp1) and breast cancer susceptibility gene 1 (BRCAI) (Roy and Banerjee 2015). p27 Kip1 has a role in the maintenance of a quiescent phase of cells and the regulation of cell proliferation during organ development (Fero, Rivkin et al. 1996). Interestingly, studies with heterozygous null mice of p27 Kip1 have revealed that it is haploinsufficient for tumor suppressor activity (Fero, Randel et al. 1998). CyclinD/Cdk4/6 promotes cell cycle progression by binding to Cip/Kip family inhibitors to prevent inhibition of cyclinE/Cdk2 complex. In addition, by binding and sequestering Cdk4/6, the INK4 inhibitors can increase the pool of free p27Kip and promote more efficient inhibition of cyclinE/Ckd2 complex (Sherr and Roberts 1995). Knock-out studies have revealed that INK4 and Cip/Kip inhibitors cooperate in tumor suppression in a tissue-specific manner (Franklin, Godfrey et al. 1998, Franklin, Godfrey et al. 2000). Interestingly, a recent study with CKI-impaired mice shows that INK4 and Cip/Kip inhibitors are necessary for the prevention of replication stress during development (Quereda, Porlan et al. 2015). As one would expect, the chromosomal region 9p21 carrying the INK4a and INK4b genes is frequently altered in many human cancers, underscoring the importance of CKIs in the anti-tumor activity of the cell (Ruas and Peters 1998). 10 REVIEW OF LITERATURE 3. DNA damage response (DDR) The DNA of a cell is constantly under stress during each round of replication. DNA damage can occur by spontaneous alterations such as deamination or depurination that occur during DNA replication or induced by reactive oxygen species (ROS). Other sources of DNA damage are environmental inducers such as ionizing radiation (IR), UV, chemical agents such as chemotherapeutic agents and virus infection. To secure genomic integrity, the cell is armed with multiple DNA repair mechanisms specific for the type of DNA damage. For instance, double stranded breaks (DSB) are repaired with non-homologous end joining (NHEJ) or homologous recombination (HR), while single stranded breaks (SSB) are repaired with single-strand break repair (SSBR). The initiation of a repair process and the induction of apoptosis when the damage cannot be repaired are orchestrated by a series of events known as the DNA damage response (DDR). DDR involves an intricate signaling network regulating recognition, timing and implementation of DNA repair, cell cycle arrest and apoptosis. Major mediators of DDR are phosphatidyl inositol 3 kinase-like protein kinases (PIKKs), ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related protein (ATR) and DNA-dependent protein kinase (DNA-PK). ATM and DNA-PK are activated by DSBs while ATR is activated by single stranded DNA (ssDNA) and DSBs (Ciccia and Elledge 2010). ATM/ATR promote DSB repair by recruiting DDR factors to damaged DNA. To achieve this, ATM phosphorylates the H2AX histone (also known as γH2AX) at Ser139 in nucleosomes, close to the DSB site. Once phosphorylated, γH2AX recruits DDR factors, including ATM, to the site of DNA damage as well as chromatin modifiers, like SWItch/Sucrose NonFermentable (SWI/SNF), which open the chromatin, giving access to DNA repair proteins (Lee, Park et al. 2010). Active ATM phosphorylates the checkpoint kinase 2 (Chk2), which phosphorylates and activates p53. Active p53 can induce DNA repair, cell cycle arrest or apoptosis by activating the expression of downstream target genes (Riley, Sontag et al. 2008). Additionally, ATM can phosphorylate the Cdk-activating phosphatase cell Cdc25A and induce its proteolytic degradation. This inhibits activation of Cdk2 and induces G1 arrest. If the replication fork stalls during S-phase, the consequent DNA damage will activate ATR, which phosphorylates and activates the Chk1 kinase. Active Chk1 phosphorylates and causes proteolysis of Cdc25A, inhibiting Cdk2/cyclin E/A, hence inhibiting DNA replication (Bartek and Lukas 2001). DSBs that occur in G2 phase activate both ATM and ATR, leading to phosphorylation of Chk-1 and -2 and Cdc25A. This inhibits Ckd1/cyclin B activation, preventing cells from entering 11 REVIEW OF LITERATURE M-phase (Bartek and Lukas 2007). This G2/M checkpoint is crucial to avoid the passage of damaged DNA to one of the dividing cells. Inhibition of G2/M arrest in the presence of DNA damage induces apoptosis. As discussed later in this thesis, viruses can take advantage of cell cycle checkpoints to induce cell cycle arrest and prevent apoptotic mechanisms. 4. Tumor suppressor p53 p53 is a tumor suppressor protein, originally discovered from research on murine cells transformed by the DNA tumor virus Simian virus 40 (SV40). It was found that the viral large T-antigen protein interacted with p53 (Lane and Crawford 1979, Linzer and Levine 1979, Finlay, Hinds et al. 1989). p53 is a stress sensor protein capable of halting the cell cycle and inducing apoptosis in response to stress signals such as DNA damage, aberrant expression of oncogenes and hypoxia, making it an efficient barrier against tumorigenesis (Giaccia and Kastan 1998, Bieging, Mello et al. 2014). p53 functions as a transcription factor, activating the transcription of target genes that regulate apoptosis, senescence, DNA repair and cell cycle arrest. The TP53 gene is mutated or lost in about 50% of all human cancers (Hollstein, Sidransky et al. 1991, Levine, Momand et al. 1991, Feki and Irminger-Finger 2004, Bouaoun, Sonkin et al. 2016). Interestingly tumor viruses have developed multiple mechanisms to inhibit p53 and to promote tumorigenesis (Sato and Tsurumi 2013). p53 consists of three functional domains that provide sequence-specific DNA binding (DNA binding domain, DBD), transcriptional activation (TAD domains) and oligomerization (tetramerization domain, TET) (Beckerman and Prives 2010). The vast majority of tumorassociated mutations in p53 are missense mutations in the DBD that disrupt the interaction of p53 with DNA and prevent transactivation of target genes (Thukral, Lu et al. 1995, Olivier, Hollstein et al. 2010). These mutants act in a dominant negative manner (Willis, Jung et al. 2004). Upon stress, p53 is phosphorylated at multiple sites, stabilized, and forms homo-tetrameric complexes via TET domain (Friedman, Chen et al. 1993). The p53 tetramers bind to specific p53-response DNA elements comprising two half-sites of the following consensus nucleotides: RRRCWWGYYY, where R stands for purine, W stands for A or T and Y stands for pyrimidine. These half-sites are separated by a spacer region of 0-13 nucleotides (el-Deiry, Kern et al. 1992). 4.1. The role of p53 in apoptosis p53 regulates both the intrinsic and extrinsic apoptotic pathways by activating the expression of pro-apoptotic genes (Haupt, Berger et al. 2003). p53 can induce intrinsic apoptosis by activating the expression of Bax or BH3-only proteins Bid, PUMA or PMA-Induced Protein (Noxa), and cause the release of cytochrome C from mitochondria as well as activation of caspases (Miyashita, 12 REVIEW OF LITERATURE Krajewski et al. 1994, Oda, Ohki et al. 2000, Nakano and Vousden 2001, Sax, Fei et al. 2002). Additionally, p53 can activate the expression of Apaf-1 and promote the formation of the apoptosome in response to stress (Soengas, Alarcon et al. 1999, Robles, Bemmels et al. 2001). Interestingly, p53 can also induce apoptosis in a transcriptionally independent manner by binding Bcl-XL and Bcl-2 proteins, leading to permeabilization of the outer mitochondrial membrane (Mihara, Erster et al. 2003, Chipuk, Kuwana et al. 2004, Tomita, Marchenko et al. 2006, Vaseva and Moll 2009). p53 can mediate the execution of the extrinsic apoptosis pathway by activating the expression and increasing the levels of the death receptors Fas and DR5 (Wu, Burns et al. 1997, Muller, Wilder et al. 1998) and inducing the expression of the death ligands FasL and TNF-related apoptosis-inducing ligand (TRAIL) (Maecker, Koumenis et al. 2000, Kuribayashi, Krigsfeld et al. 2008). 4.2. The role of p53 in cell cycle arrest To be able to preserve the integrity of the genome upon DNA damage, p53 can stop the cell cycle at the G1 or G2 phase to prevent replication of the damaged DNA in S-phase or segregation of the damaged chromosomes during mitosis (Giono and Manfredi 2006). p53 can activate the expression of the p21Cip1 gene cyclin dependent kinase inhibitor 1A (CDKN1A) upon DNA damage (e.g. caused by reactive oxygen species or radiation) and induce G1 cell cycle arrest (el-Deiry, Tokino et al. 1993). Furthermore, p53 can induce G2/M cell cycle arrest mainly by activating genes, such as CDKN1A, Growth Arrest and DNA Damage (GADD45), 14-3-3δ and Reprimo, whose products inhibit the Cdk1/cyclin B complex. p53 can also repress Cdk1 and cyclin B expression directly (Taylor and Stark 2001). 4.3. Modulation of p53 activity A classical p53 response consists of a sequence of events: i) stress-induced stabilization of p53 by phosphorylation ii) tetramerization and binding of p53 to DNA and iii) target gene activation in collaboration with other components of the DNA transcription machinery. The activity of p53 is regulated by multiple different post-transcriptional modifications including phosphorylation, acetylation and ubiquitination. These modifications have an effect on the stability, transcriptional function and cellular localization of p53. Depending on the type of stress or form of DNA damage, certain types of kinases are activated and phosphorylate p53. For instance, ATM, ATR, DNA-PK, Chk1 and Chk2 kinases phosphorylate and activate p53 upon DNA damage (Kruse and Gu 2009). These kinases phosphorylate p53 at Ser 15 and Ser 20. These phosphorylation events lead to the stabilization of p53 by disrupting the 13 REVIEW OF LITERATURE interaction between p53 and its negative regulator, the E3 ubiquitin ligase, Mdm2 (Shieh, Ikeda et al. 1997, Shieh, Ahn et al. 2000, Appella and Anderson 2001). Acetylation of p53 is important for its transactivation function and for the recruitment of cofactors (Kruse and Gu 2009). p53 is acetylated mainly by the histone acetyltransferase (HAT) CREB binding protein (CBP/p300) (Gu and Roeder 1997). During the stress response, the acetylation levels of p53 increase rapidly and this correlates with the activation and stabilization of p53 (Ito, Lai et al. 2001). p53 can be acetylated at multiple different lysine residues, which modulates the recruitment of different cofactors on target gene promoters, thus regulating the type of p53 response (Kruse and Gu 2009). For example, K120 acetylation of p53 is required for transactivation of the pro-apoptotic Bax and Puma genes, but not for p21CIP1 or Mdm2 expression (Tang, Luo et al. 2006, Mellert, Sykes et al. 2007). Under normal conditions, p53 is constantly expressed, but its half-life is only 5-20 minutes since it is continuously ubiquitinated mainly by Mdm2 and degraded by the proteasome. MDM2 gene expression is, in turn, regulated by p53, thus establishing a negative feedback loop which maintains low levels of p53 in normal, unstressed cells (Li, Brooks et al. 2003, Chao 2014). 4.4. Regulation of p53 by Mdm2 Mouse double minute-2 gene (MDM2) was firstly found as name denotes from double minute chromosomes of spontaneously transformed balb/c3T3 mouse cells (Cahilly-Snyder, Yang-Feng et al. 1987) and later the human homolog was found to be associated with tumor suppressor p53 (Momand, Zambetti et al. 1992). Mdm2 consists of several domains, which are important for its function. In NH2-termini locates p53 binding domain and in central region locates nuclear localization sequence (NLS), nuclear export signal (NES) and acidic domain and in COOH-termini locates RING finger domain carrying E3 ubiquitin ligase activity (Fakharzadeh, Trusko et al. 1991, Chen, Marechal et al. 1993, Roth, Dobbelstein et al. 1998, Fang, Jensen et al. 2000). Mdm2 can inhibit p53 function by two means either by binding transactivation domain of p53 and thus blocking its ability to activate transcription of its target genes or target p53 for proteasomal degradation (Kussie, Gorina et al. 1996). Depending on Mdm2 levels and stress conditions Mdm2 can either monoubiquitinate or polyubiquitinate p53 in the nucleus. Hence in normal unstressed conditions when there is a low level of Mdm2 it binds and monoubiquitinates p53 and promotes nuclear export to keep p53 away from its transcriptional targets. In stressed conditions when there is a high amount of Mdm2 it can in collaboration with transcriptional co-activator p300 polyubiquitinate p53 and induce 26S proteasome degradation of p53 in the nucleus (Lohrum, 14 REVIEW OF LITERATURE Woods et al. 2001, Grossman, Deato et al. 2003, Li, Brooks et al. 2003). It has been shown that monoubiquitination will expose COOH-terminal NES sequence of p53 and allow proper nuclear export and in the cytoplasm where p53 can be further poly-ubiquitinated by other E3 and E4 ligases like for example ubiquitination factor E4B (UBE4B) and directed to 26S proteasome for degradation (Geyer, Yu et al. 2000, Nie, Sasaki et al. 2007, Wu, Pomeroy et al. 2011). Alternatively cytoplasmic monoubiquitinated p53 is transported to mitochondrial membrane where it is deubiquitinated by herpesvirus-associated ubiquitin-specific protease (HAUSP) and protected from the degradation and thus promoting apoptosis (Marchenko, Wolff et al. 2007). Indicating transcription independent function of p53 and underlining the important role of Mdm2 levels in control of p53 activity. 4.5. Regulation of Mdm2 Mdm2 protein stability and function is regulated by several stress signals such as oncogenic activation or DNA damage, which activates multiple cellular pathways controlling posttranscriptional modifications and cellular localization of Mdm2 and thus Mdm2-p53 interaction. Aberrant mitogenic signaling operated by oncogenes such as Ras and V-Myc Avian Myelocytomatosis Viral Oncogene Homolog (c-Myc) interfere Mdm2 function and stabilize and activates p53. This process is conducted by another tumor suppressor protein p14ARF, which is negative regulator of Mdm2 protein (Palmero, Pantoja et al. 1998, Zindy, Eischen et al. 1998). p14ARF gene is transcribed from alternate reading frame of INK4A/ARF gene locus expressing in addition to p14ARF cyclin D dependent inhibitor: p16INK4A (Mao, Merlo et al. 1995). p14ARF inhibit E3 ubiquitin ligase activity of Mdm2 (Pomerantz, Schreiber-Agus et al. 1998, Honda and Yasuda 1999). And it has been shown that p14ARF is nucleolar protein is binding central domain of Mdm2 and sequester Mdm2 to nucleolus thus stabilizing nucleoplasmic p53 (Weber, Taylor et al. 1999, Weber, Kuo et al. 2000). Another important regulator of Mdm2 and p53 is MdmX or also known as Mdm4 protein which is a homologue of Mdm2 and negative regulator of p53. However MdmX lacks E3 ubiquitin ligase activity towards p53 it can inhibit p53 function by preventing its transcriptional activity (Toledo, Krummel et al. 2006). MdmX can regulate protein stability and activity of Mdm2 by forming hetorodimers with Mdm2 via their RING finger domains and stabilize Mdm2 by preventing autoubiquitination and enhancing its E3 ligase activity towards p53 (Linares, Hengstermann et al. 2003, Wang, Wang et al. 2011). 15 REVIEW OF LITERATURE 5. Kaposi’s sarcoma herpesvirus (KSHV) Kaposi’s sarcoma herpesvirus (KSHV or HHV-8) is a human gamma2-herpesvirus and the causative agent of three human cancers: Kaposi’s sarcoma (KS) and two lymphoproliferative malignancies: primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). 5.1. Discovery of KSHV and its associated malignancies Kaposi’s sarcoma (KS) was originally identified by the Hungarian dermatologist Moritz Kaposi in 1872 (Kaposi 1872). During the AIDS epidemic in the early 1980s, KS cases increased dramatically and the correlation between AIDS and KS led to the hypothesis that another infectious agent would be involved in the development of KS (Beral, Peterman et al. 1990). Finally, in 1994, Moore and Chang were able to extract and identify KSHV DNA sequences from KS lesions of AIDS patients (Chang, Cesarman et al. 1994) A year later Cesarman et al also found KSHV DNA sequences from AIDS patients suffering from body cavity-based lymphoma (BCBL, also known as PEL) (Cesarman, Chang et al. 1995) and established the BCBL-1 cell line stably infected with KSHV. This cell line has become an instrumental tool for the further development of the KSHV research field (Cesarman, Moore et al. 1995, Arvanitakis, Mesri et al. 1996). Around the same time when causality between KSHV and PEL was found, another lymphoproliferative disorder, plasmablastic variant of MCD, was found to be associated with KSHV infection (Soulier, Grollet et al. 1995, Gessain, Sudaka et al. 1996). 5.1.1. Kaposi’s sarcoma KS usually appears as highly angiogenic lesions on the skin of the upper and lower body extremities or on mucosal surfaces. Visceral organs and lymph nodes can also harbor these lesions. Histologically, KS lesions consist of spindle-shaped tumor cells, with a high number of disorganized and leaky blood vessels and extravasated red blood cells causing the purple, red or brown coloration of the lesion (Antman and Chang 2000, Orenstein 2008). KS lesions are not like typical tumor tissues arising from outgrowth of a clonal cell population. They instead consist of a wide variety of different cell types such as endothelial cells, macrophages and smooth muscle cells (Weich, Salahuddin et al. 1991, Kaaya, Parravicini et al. 1995, Sturzl, Brandstetter et al. 1995, Dupin, Fisher et al. 1999). Although KS spindle cells have been identified to be of endothelial origin and express multiple blood endothelial markers (like CD31 and CD34) it has been reported that these cells can also express lymphatic endothelial markers like vascular growth factor receptor 3 (VEGFR3) and Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1) (Browning, 16 REVIEW OF LITERATURE Sechler et al. 1994, Weninger, Partanen et al. 1999, Hong, Foreman et al. 2004, Wang, Trotter et al. 2004). KS lesions progress through different stages. In the early stage, also called the patch and plaque stage, lesions appear small and flat. In the later, nodular stage, lesions grow further in thickness and they contain highly proliferating spindle cell layers and slit-like neovascular spaces (Ganem 2010). Over 95% of KS lesions contain KSHV DNA (Dupin, Fisher et al. 1999). There are four forms of KS: Classic KS, Endemic KS, Iatrogenic/post-transplant KS and AIDS-associated/Epidemic KS. The classic KS is usually an indolent and chronic disease affecting elderly men of Mediterranean and eastern European Jewish ancestry (Oettle 1962). Endemic KS mainly affects young populations of sub-Saharan Africa, where it is one of the most common cancers. Endemic KS is not associated with human immunodeficiency virus (HIV) infection, but it is more aggressive than classic KS (Taylor, Templeton et al. 1971, Bayley 1984). The Iatrogenic KS affects patients treated with immunosuppressive therapy after organ transplantation (Andreoni, Goletti et al. 2001, Marcelin, Calvez et al. 2007). The AIDS-associated KS, also called epidemic KS, is the most common and most aggressive form of KS affecting HIV-positive individuals, and it has been identified as an AIDS-defining disease (Giffin and Damania 2014). Recent epidemiological studies indicates that although combination antiretroviral treatment (cART) is able to control HIV-1 replication and restore CD4+ T-cell counts of HIV positive individuals and thus KS incidences have been significantly declined, KS is the most common cancer in HIV infected individuals and is the most prevalent cancers in Africa (Casper 2011, Rohner, Valeri et al. 2014, Labo, Miley et al. 2015). 5.1.2. Primary effusion lymphoma Primary effusion lymphoma (PEL) is a non-Hodgkin’s lymphoma (NHL) consisting of monoclonal B-type cells that expand in body cavities such as the pleural space, pericardium and peritoneum. There are also some solid PEL cases, where the tumors usually appear extra-nodaly (Carbone, Gloghini et al. 2005). Each tumor cell has about 30-100 copies of the KSHV genome (Renne, Lagunoff et al. 1996). Clonal immunoglobulin gene rearrangements and somatic hypermutations have been observed in PEL cells, indicating that these cells originate from the post-germinal center B-cells (Matolcsy, Nador et al. 1998). PEL is not a typical lymphoma, since it lacks c-myc gene rearrangements, Bcl-2, Ras and, in most cases, TP53 gene alterations (Cesarman and Knowles 1999). PEL cells lack expression of B- and T-cell associated antigens but they express the CD45 antigen, which is a marker of haematolymphoid cells. Additionally PEL cells express various markers of lymphocyte activation such as CD30, CD38, CD71 and several plasma cell markers including CD138, VS38c and MUM1/IRF4 (Cesarman and Knowles 1999, Carbone, Gloghini et al. 17 REVIEW OF LITERATURE 2001). All of this indicates that PEL cells partially resemble differentiated plasma cells. PEL is a rare disease, being present in only 4% of HIV-associated NHL cases and 0,4 % of HIV-unrelated NHLs (Carbone, Gloghini et al. 1996). PEL cases are rarely found in HIV-negative patients who have received a solid organ transplant or in elderly men of Mediterranean descent (Jones, Ballestas et al. 1998, Dotti, Fiocchi et al. 1999, Klepfish, Sarid et al. 2001). PEL has a poor prognosis and the median survival time after diagnosis is only 6 months (Boulanger, Gerard et al. 2005). Prognosis of PEL in HIV-infected individuals is dependent on highly active antiretroviral therapy (HAART) and performance status prior to PEL diagnosis (Simonelli, Spina et al. 2003, Boulanger, Gerard et al. 2005). Additionally, KSHV viral load might be a prognostic factor for the outcome of PEL disease (Simonelli, Tedeschi et al. 2006). Currently there is no effective therapy for PEL and conventional chemotherapy is not curative. Therefore, novel therapeutic approaches are needed. 5.1.3. Other KSHV associated diseases KSHV is associated with a plasmablastic variant of multicentric Castleman’s disease (MCD) (Soulier, Grollet et al. 1995). MCD is a rare neoplastic lymphoproliferative disorder comprising highly proliferating IgM λ-restricted plasmablasts in the mantle zone of the germinal center (Du, Liu et al. 2001). Almost all HIV-positive MCD patients are co-infected with KSHV and 40% of HIV-negative MCD patients are KSHV-positive. MCD patients who are co-infected with HIV and KSHV have a more aggressive disease with poorer prognosis and they frequently have other malignancies, including KS and PEL (Soulier, Grollet et al. 1995). MCD patients have systemic lymphadenopathy and inflammatory manifestation and disease progression is caused by dysregulated IL-6, IL-10 and vascular growth factor (VEGF) cytokine production. KSHV can further promote the progression of MCD by expressing viral IL-6 (vIL6), which can increase the expression of human IL-6 and VEGF (Giffin and Damania 2014). High viral loads of KSHV and high levels of vIL6 expression have been linked to rapid progression of the disease (Parravicini, Corbellino et al. 1997, Oksenhendler, Carcelain et al. 2000). KSHV infection is also linked to a more recently identified disease called KSHV-associated inflammatory cytokine syndrome (KICS). In this syndrome patients have MCD-like inflammatory symptoms, but without lymphadenopathy. KICS patients have elevated cytokine levels of IL-6, IL10, C-reactive protein and vIL6 (Uldrick, Wang et al. 2010). These patients have high KSHV viral loads, indicating a high rate of lytic KSHV infection (Polizzotto, Uldrick et al. 2012). 18 REVIEW OF LITERATURE 5.2. KSHV biology 5.2.1. Taxonomy KSHV belongs to the large herpesviridae family of double-stranded DNA viruses. The currently known eight human herpesviruses (HHV) can be categorized into three subgroups: α-, β- and γherpesviruses. Herpes simplex-1 and 2 and varicella zoster (VZV or HHV-3) viruses belong to the α-herpesvirus group. The β-herpesvirus group comprises cytomegalovirus (CMV or HHV-5), HHV6 and 7 viruses. The γ-herpesvirus group is divided into two subgroups: γ-1lymphocryptoviruses, including Epstein-Barr virus (EBV or HHV-4), and γ-2 rhadinoviruses, including KSHV or HHV-8 (Giffin and Damania 2014). EBV infection causes mononucleosis, and like KSHV, it is the causative agent of several human cancers in immunocompromised individuals (Cesarman 2011). 5.2.2. Viral transmission and infection rates KSHV can be transmitted via saliva, and in endemic countries, mother-to-child transmission is fairly common (Moore 2000). Additionally, KSHV can be transmitted via sexual contact or blood transfusions (Martin, Ganem et al. 1998, Hladik, Dollard et al. 2006). There is a high global variation in seroprevalence of KSHV, since in the endemic areas, such as in Africa, seroprevalence is as high as over 50 %, and in Southern Europe it varies between 10-30 %, whereas in Northern Europe and in the United States it is only 1-6 % (Gao, Kingsley et al. 1996, Kedes, Operskalski et al. 1996, Simpson, Schulz et al. 1996, Schatz, Monini et al. 2001). Usually KSHV DNA genomes are detected in peripheral B-cells, but also in T-cells, endothelial cells, epithelial cells and even monocytes (Ambroziak, Blackbourn et al. 1995, Blasig, Zietz et al. 1997, Dupin, Fisher et al. 1999, Henry, Uthman et al. 1999, Pauk, Huang et al. 2000). 5.2.3. Virion structure and viral entry KSHV virion consists of lipid bilayer envelope, tegument, icosahedral capsid structures and linear double stranded DNA genome (Figure 3A). Transmembrane glycoproteins of the virion envelope mediate attachment and entry of the KSHV virion via fusion or endocytosis into target cell (Akula, Pramod et al. 2001, Pertel 2002). KSHV glycoproteins K8.1A and gB bind heparin sulfate of the target cell and mediate attachment of the virion to the target cell membrane (Akula, Pramod et al. 2001, Birkmann, Mahr et al. 2001, Wang, Akula et al. 2001). KSHV virion entry occurs via interaction between viral glycoproteins and target cell surface receptors and currently it is known that integrins α3β1, αVβ3 and αVβ5 and 12-transmembrane transporter protein xCT plays a role in this process (Akula, Pramod et al. 2002, Wang, Akula et al. 2003, Kaleeba and Berger 2006, Garrigues, Rubinchikova et al. 2008). KSHV virion enters the cell via clathrin mediated endocytosis 19 REVIEW OF LITERATURE or micropinocytosis (Akula, Naranatt et al. 2003, Raghu, Sharma-Walia et al. 2009). Tegument layer of the virion exists between the envelope and capsid and it consist of viral proteins such as open reading frames (ORFs) 45 and 64 and 11 viral RNA transcripts (Bechtel, Grundhoff et al. 2005, Bechtel, Winant et al. 2005). Icosahedral KSHV capsid forms a shell which is composed of capsomers and capsid floor compartments which are composed of major capsid proteins ORF25, ORF62, ORF26, ORF17.5 and minor capsid protein ORF65 (Wu, Lo et al. 2000, Nealon, Newcomb et al. 2001). KSHV genome is linear double stranded DNA surrounded by capsid. Genome consist of 140 kb long coding sequence flanked by terminal repeat (TR) regions (Renne, Lagunoff et al. 1996). KSHV genome encodes 87 ORFs and also mRNA and other non-coding RNAs (Figure 3B) (Russo, Bohenzky et al. 1996, Samols, Hu et al. 2005). One of the striking features of the KSHV genome is that it contains a high number of ORFs which are human orthologues which play an important role in tumorigenesis. Figure 3. Structure of KSHV virion and genome. A) KSHV virion consists of lipid bilayer envelope, tegument, icosahedral capsid structures and linear double stranded DNA genome. B) KSHV genome consist of 140 kb long coding sequence flanked by terminal repeat (TR) regions KSHV genome encodes 87 ORFs and also microRNAs (miRNA 20 REVIEW OF LITERATURE cluster). During latency, KSHV expresses only a small number of its genes and miRNAs (Latency locus, green arrows), whereas during lytic replication all lytic genes are expressed (red arrows). 5.2.4. KSHV life cycle KSHV establishes persistent life-long infection and manipulates host cell proliferation, differentiation, metabolism, apoptosis and immunity (Mesri, Cesarman et al. 2010). KSHV has two life cycle phases: the latent phase and the lytic replication phase. Latency is a silent mode of infection whereby KSHV hides its DNA genome inside the host cell nucleus, only synthesizing a few proteins and miRNAs, thus avoiding detection by the immune system. In the lytic replication phase, new viral progeny are produced, ultimately leading to the lysis of the host cell. Latency is the default infection mode for KSHV (Zhong, Wang et al. 1996). Physiological factors like hypoxia, oxidative stress, and inflammatory cytokines or chemicals such as the protein kinase C (PKC) antagonist 12-O-tetradecanoylphorbol-13-acetate (TPA), or HDAC inhibitors such as sodium butyrate (NaB) can reactivate the virus from its latent phase and initiate the lytic replication phase (Yu, Black et al. 1999, Davis, Rinderknecht et al. 2001, Pantry and Medveczky 2009, Ye, Zhou et al. 2011). 5.2.5. Establishment of latent infection Upon entry into its target cell, KSHV activates the PI3K signaling pathway-activating Rho GTPases to alter the actin cytoskeleton and aid in the delivery of the capsid into the cytoplasm of the infected cell (Sharma-Walia, Naranatt et al. 2004, Greene and Gao 2009). The nucleocapsids travel to the nucleus and after uncoating, release the viral genome (Naranatt, Akula et al. 2003, Naranatt, Krishnan et al. 2005). The released linear genome is circularized and associates with cellular histones (i.e. it becomes 'chromatinized') forming a tightly packed viral episome. The chromatinization process protects the episome from DNA damage and enables transcriptional regulation of viral gene expression (Lieberman 2013). In this state, the viral episome is then tethered to chromosomes of the host cell (extrachromosomal episome) to ensure its maintenance during cell divisions (Ballestas, Chatis et al. 1999). During latency, KSHV expresses only a small number of its genes (Table 1): the Latency Associated Nuclear Antigen (LANA and LANA2 also called viral interferon regulator factor 3 (vIRF3)), viral cyclin (v-cyclin), viral FLIP (vFLIP), Kaposin A and B (Fakhari and Dittmer 2002). KSHV genome carries also viral miRNAs. miRNAs are short non-coding RNAs (20-24 nucleotides) that modulate gene expression by binding 3´Untranslated region (3’UTR) of the target gene. 5’end of the miRNA carry 2-8 nucleotides long seed sequence which determines the target for 21 REVIEW OF LITERATURE the miRNA (Bartel 2009). KSHV encodes 12 pre-miRNAs which are located in the latency locus. Ten of these miRNAs (miR-K12-1 to miR-K12-9 and miR-K12-11) are located in the intron of the K12 gene and two of them (miR-K12-10 and miR-K12-12) are located in the coding region of K12 gene (Figure 3B) (Zhu, Haecker et al. 2013). Many of the KSHV miRNAs have been reported to target cellular genes regulating proliferation, cell survival and apoptosis (Haecker, Gay et al. 2012, Zhu, Haecker et al. 2013). During latency, the KSHV genome is replicated by the host DNA replication machinery along with cellular DNA once every cell division. After mitosis, duplicated viral genomes are segregated amongst daughter cells, but no viral progeny are produced (Cotter and Robertson 1999, Barbera, Chodaparambil et al. 2006, You, Srinivasan et al. 2006, Matsumura, Persson et al. 2010). Establishment and maintenance of latent infection is inefficient in vitro, since cells extracted from KS lesions or de novo-infected cell lines tend to lose their KSHV genome upon cell culturing (Dictor, Rambech et al. 1996, Lagunoff, Bechtel et al. 2002) . Only exceptionally do cell lines extracted from PEL patients carry KSHV genomes stably in culture (Cesarman, Moore et al. 1995). LANA is the hallmark of KSHV latent infection. LANA can be visualized with indirect immunofluorescence staining as a speckled nuclear pattern (Gao, Kingsley et al. 1996) . Nearly all cells in KS lesions express LANA (Dittmer, Lagunoff et al. 1998) and LANA protein is detected in all KSHV-associated malignant cells (Dupin, Fisher et al. 1999). LANA is encoded by the ORF73 gene (Kedes, Lagunoff et al. 1997, Kellam, Boshoff et al. 1997, Rainbow, Platt et al. 1997) and is transcribed from the polycistronic latency transcript locus together with v-cyclin (ORF72) and vFLIP (ORF71) (Dittmer, Lagunoff et al. 1998). LANA’s most important functions are to establish and maintain stable latent infection by ensuring replication and segregation of the KSHV genome during cell division and to circularize the KSHV genome by binding terminal repeat (TR) regions (Ballestas, Chatis et al. 1999, Ballestas and Kaye 2001). 5.2.6. Reactivation from latency and the lytic cycle The term, “reactivation” refers to the induction of the lytic replication phase, which can be triggered by a variety of stresses. Transcription of the viral lytic genes is activated in a carefully orchestrated cascade. The first viral lytic gene synthesized upon reactivation is the Replication and Transcription Activator (RTA). RTA is encoded by the gene, ORF50, and is necessary and sufficient to disrupt latency and initiate a full lytic cascade (Lukac, Renne et al. 1998, Sun, Lin et al. 1998). RTA is a transcription factor responsible for the transactivation of the downstream lytic genes and autoactivation of its own promoter (Guito and Lukac 2012). During the lytic cycle, viral genes are expressed in a temporally regulated order with immediate early (IE) genes expressed first, followed 22 REVIEW OF LITERATURE by expression of delayed-early (DE) genes that trigger lytic DNA replication, followed by the expression of late (L) genes that encode the structural proteins of the virion (Sun, Lin et al. 1999). RTA can activate the expression of multiple DE genes directly or indirectly, such as K2 encoding viral interleukin-6 (vIL-6), polyadenylated nuclear RNA (PAN), ORF57 encoding mRNA transport and accumulation protein (MTA), K8 encoding replication-associated protein (RAP), ORF74 encoding viral G protein-coupled receptor (vGPCR) and many others (Table 1) (Purushothaman, Uppal et al. 2015). The RTA responsive elements, RRE, mediate direct binding to the respective promoters (Song, Deng et al. 2003). RTA can also mediate gene expression indirectly by interacting with cellular transcription factors (Figure 4) (Liang, Chang et al. 2002, Song, Deng et al. 2003, Wang, Wu et al. 2003) such as the Notch signaling mediator Recombining binding protein suppressor of hairless (RBP-Jκ). Interestingly, virus reactivation was inhibited in KSHV-infected RBP-Jκ-null murine fibroblasts, underscoring the important role of RBP-Jκ for reactivation in vivo (Liang and Ganem 2003). Additionally, RTA interacts with other cellular TFs such as AP-1, a heterodimeric transcription factor complex consisting of proteins belonging to the c-Jun and c-Fos families (Hess, Angel et al. 2004) that co-operate with RTA and activate the expression of some lytic genes, (Byun, Gwack et al. 2002, Wang, Wu et al. 2004) thereby promoting the lytic gene expression cascade (Wang, Wu et al. 2003). Also, the X-box binding protein (XBP-1) has been shown to transactivate ORF50 and induce reactivation of PEL cells (Wilson, Tsao et al. 2007, Yu, Feng et al. 2007). In addition to cellular TFs, RTA activity requires the recruitment of transcriptional co-factors like the histone acetyl transferase CREB binding protein (CBP), the chromatin remodeling complex SWI/SNF and the co-activator TRAP/mediator (Figure 4) (Gwack, Baek et al. 2003). The activity of RTA can also be repressed by some cellular proteins. Interferon regulatory factor (IRF)-7 can repress RTA-mediated transactivation by competing with RTA for ORF57 promoter binding (Wang, Zhang et al. 2005). As a countermeasure, RTA has E3 ubiquitin ligase activity against IRF-7 which targets the cellular proteins for proteasomal degradation, thereby counteracting host defense mechanisms and ensuring viral reactivation (Yu, Wang et al. 2005). 23 REVIEW OF LITERATURE Table 1. Classification of KSHV genes Gene and gene description Life cycle phase ORF73/LANA (Latency-Associated Nuclear Antigen) Latent K10.5/LANA2/vIRF3 (viral interferon regulatory factor 3) Latent ORF72/v-cyclin (viral homolog of human cyclin D2 Latent ORF71/vFLIP (viral FLIP) Latent K12/Kaposin Latent miRNA cluster (12 viral pre-miRNAs) Latent ORF50/RTA (Replication and Transcription Activator) Lytic (IE) PAN (polyadenylated nuclear RNA) Lytic (DE) ORF57/MTA (mRNA transport and accumulation protein) Lytic (DE) K8/RAP/K-bZIP (replication-associated protein, transcription Lytic (DE) factor) ORF74/vGPCR (viral G protein-coupled receptor Lytic (DE) K2/vIL6 (viral interleukin-6) Lytic (DE) K9/vIRF1 (viral interferon regulatory factor 1) Lytic (DE) K10/vIRF4 (viral interferon regulatory factor 4) Lytic (DE) ORF16/vBcl2 (viral Bcl-2) Lytic (DE) K7/vIAP (anti-apoptotic protein) Lytic (DE) ORF22 (glycoprotein) Lytic (L) ORF17.5 (major capsid protein) Lytic (L) ORF25 (major capsid protein) Lytic (L) ORF26 (major capsid protein) Lytic (L) ORF62 (major capsid protein) Lytic (L) ORF 65 (minor capsid protein) Lytic (L) ORF45 (tegument protein) Lytic (L) ORF64 (tegument protein) Lytic (L) K8.1A (glycoprotein) Lytic (L) 24 REVIEW OF LITERATURE 5.2.7. Regulation of reactivation 5.2.7.1. Chromatin modifications controlling the KSHV life cycle Chromatin consists of DNA wrapped around histone proteins (H2A, H2B, H3 and H4) that associate together forming an octameric complex: the nucleosome. The structure of chromatin, i.e. the degree of condensation of DNA and nucleosomes, can be modulated by post-translational modification of the N-terminal tails of the core histones. These structural changes determine whether or not the double helix can be accessed by transcription or DNA replication factors i.e. low (euchromatin) or high condensation (heterochromatin), respectively (Bannister and Kouzarides 2011). Histones can be acetylated, methylated, phosphorylated or ubiquitinated and these modifications affect chromatin condensation. Histone modifying enzymes such as HATs, HDACs, histone methyl transferases (HMTs) and histone demethylases are among the enzymes that perform post-translational modifications of the histones. Modified histones serve as platforms for the recruitment of both activators and repressors of transcription. Thus, histone modifying enzymes affect gene expression (Bannister and Kouzarides 2011). Similar to cellular nucleosomes, the histones in the chromatinized KSHV episome are also modified to regulate transcription of viral genes and thus the viral life cycle (Figure 4). During latency, the promoters of latent genes are associated with histones that have activating modifications such as H3K27me3 (Gunther and Grundhoff 2010, Toth, Maglinte et al. 2010), while the early lytic genes ORF50 (RTA) and ORF48 have bivalent histone modifications, including both repressive marks (H3K27me3) and active marks (H3K4me3). Conceivably, the balance between these modifications determines the transcriptional state of these genes and allows viral gene expression to switch from latent to lytic. As mentioned earlier, the KSHV-infected cells are mostly in the latent phase. Thus, during this phase, the promoters of the late lytic genes contain repressive chromatin marks (e.g. H3K27me3 and H3K9me3), providing the transcriptional control needed to maintain latency. Upon reactivation of lytic genes, the levels of active histone marks, such as AcH3 and H3K4me3, increase, while those of repressive modifications, e.g. H3K27me3, decrease. This results in the upregulation of viral lytic gene transcription (Toth, Maglinte et al. 2010). Similarly, histone methyltransferase enzymes belonging to the Polycomb repressive complex 2 (PRC2) EZH2, which are responsible for the repressive H3K27me3 marks during latency, dissociate from the reactivated KSHV genome, favoring activation of IE and DE gene promoters (Figure 4). This de-repression mechanism is crucial for the maintenance of latency, as inhibition of EZH2 leads to expression of 25 REVIEW OF LITERATURE lytic genes even during latency in latent PEL cells, showing that the PRC2 complex is repressing expression of lytic genes in latency (Toth, Maglinte et al. 2010). Furthermore, overexpression of demethylases of H3K27me3, Ubiquitously transcribed tetratricopeptide repeat gene on X chromosome (UTX) and Jumonji domain containing-3 (JMJD3) cause induction of lytic gene expression in the latent cells (Gunther and Grundhoff 2010, Toth, Maglinte et al. 2010). It has been shown that demethylases UTX and JMJD3, and H3K4me3 histone methyltransferase histone-lysine N-methyltransferase 2 (MLL2) are recruited to the ORF50 promoter by PAN RNA to activate expression of IE genes (Figure 4) (Rossetto and Pari 2012). This could suggest a PAN RNA-driven positive feedback mechanism to boost expression of RTA and other IE genes in the late lytic reactivation phase. More recently, it has been reported that during latency, the RTA promoter is repressed by the transcriptional repressors Hey1 and mSin3a. Upon reactivation, the E3 ubiquitin ligase activity of RTA targets these cellular proteins for degradation, making the reactivation process more efficient (Gould, Harrison et al. 2009). Figure 4. Regulation of KSHV reactivation. During latency transcription of lytic genes is repressed by HDAC and by histone methyltransferase enzymes belonging to the PRC2 complex. Physiological factors such as hypoxia, ROS or 26 REVIEW OF LITERATURE chemicals like TPA, HDAC inhibitors can reactivate latent infection. Upon reactivation the levels of active histone marks, such as acetylated histone H3 (AcH3) and trimethylated histone 3 at lysine 4 (H3K4me3) increase. Histone demethylases UTX and JMJD3 and MLL and transcriptional co-factors like the histone acetyl transferase CBP and the chromatin remodeling complex SWI/SNF open the viral chromatin. KSHV replication and transcription activator (RTA/ORF50) activates transcription of lytic genes directly and in co-operation with transcription factors XBP-1, AP-1) and RBP-Jκ. Lytic reactivation leads to production of new viral progeny and ultimately leading to the lysis of the host cell. 5.2.7.2. Chemical induction The KSHV lytic cycle can be artificially induced by several chemicals. These include the PKC agonist, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), pan inhibitors of HDACs such as NaB and inhibitors of DNA-methyl transferase (DNMTs). Chemical induction can significantly enhance reactivation in cell culture systems, offering a valuable tool to study the mechanisms of lytic replication in vitro. 5.2.7.2.1. Phorbol ester TPA TPA is a protein kinase C agonist (Niedel, Kuhn et al. 1983), and induces the full lytic cycle of KSHV in a PKC-delta-dependent manner (Renne, Zhong et al. 1996). Importantly, TPA-induced reactivation can be enhanced by pre-synchronizing cells in the S-phase (McAllister, Hansen et al. 2005). TPA also activates JNK, a kinase that phosphorylates and activates the DNA binding domain of the transcription factor c-Jun (Engedal, Korkmaz et al. 2002, Yamasaki, Takahashi et al. 2009). It has also been shown that TPA enhances the binding of AP-1 to the ORF50 promoter and increases the phosphorylation and activation of c-Jun (Wang, Wu et al. 2004). These are among the few characterized mechanisms that could explain how TPA treatments trigger KSHV reactivation. 5.2.7.2.2. HDAC inhibitors The most commonly used HDAC inhibitor to study KSHV reactivation and the lytic cycle is NaB, which is a broad-spectrum class I HDAC inhibitor (Candido, Reeves et al. 1978). NaB treatment induces expression of RTA, leading to the full activation of the lytic cycle (Miller, Heston et al. 1997, Yu, Black et al. 1999). As one would expect, during latency, the promoter of ORF50 is associated with several HDACs (HDAC 1, 5 and 7) that keep it repressed. By increasing the acetylation levels of the core histones H3 and H4, NaB induces the de-repression of ORF50 (Lu, Zhou et al. 2003, Toth, Maglinte et al. 2010). This is also accompanied by the recruitment of several transcription factors that bind to 'open' chromatin, such as Sp1, CBP HAT and the chromatin remodeling complex SWI/SNF (Lu, Zhou et al. 2003, Ye, Shedd et al. 2005). To reveal which of the HDACs are regulating the lytic cycle, researchers in Lucak's lab tested several class I and II 27 REVIEW OF LITERATURE HDAC inhibitors, including NaB, valproic acid (VPA) and trichostatin A (TSA) (Shin, DeCotiis et al. 2014). This study revealed that VPA, which is a class I and II HDAC inhibitor, was the most potent inducer of reactivation. On the contrary, overexpression of HDAC 1, 3 and 6 inhibited reactivation of PEL cells even in the presence of TPA (Shin, DeCotiis et al. 2014), indicating that histone modifications are downstream of TPA signaling and that they are crucial for the regulation of KSHV reactivation. Moreover, a recent report shows that the class III HDAC inhibitors nicotinamide (NAM) and sirtinol could also induce reactivation of latent PEL cells (Li, He et al. 2014). Similarly to NaB, these HDAC inhibitors also change the chromatin landscape of the ORF50 promoter from the inhibited to active state (Li, He et al. 2014). 5.2.7.3. Natural inducers of reactivation 5.2.7.3.1. Hypoxia Cells respond to low oxygen levels, i.e. hypoxia, by upregulating the hypoxia-inducible factor (HIF), a transcription factor that binds and activates the hypoxia response element (HRE) on the promoter of target genes (Wang, Jiang et al. 1995). It has been shown that KSHV infection induces HIF1α and HIF2α expression in endothelial cells (Carroll, Bu et al. 2006). Furthermore, hypoxic cell culture conditions induce reactivation of PEL cells (Davis, Rinderknecht et al. 2001). Analysis of the ORF50 promoter revealed that it contains a DNA sequence similar to a HRE (Haque, Davis et al. 2003). Interestingly, it has been shown that LANA, a latent protein that normally prevents reactivation, interacts with HIF1α in the hypoxic PEL cells and enhances HIF1α-induced expression of RTA (Cai, Lan et al. 2006). Recent studies have identified HREs in promoters of both LANA and RTA, and demonstrated that HIF1α and HIF2α induce LANA expression (Veeranna, Haque et al. 2012). This study revealed that RTA interacts with HIF1α in the hypoxic PEL cells, and that hypoxia induced RTA-mediated induction of LANA expression (Veeranna, Haque et al. 2012). These studies suggest the existence of a complex positive-feedback loop whereby hypoxia induces the expression of LANA and RTA, which in turn enhance their own expression and control reactivation. In addition to HIF1α, it has also been shown that hypoxia activates XBP-1, which upregulates the expression of ORF50 and induces reactivation (Dalton-Griffin, Wilson et al. 2009). Finally, the role of hypoxia in virus reactivation was confirmed by recent studies showing that genetic depletion of the hypoxia-sensitive transcriptional repressor KRAB-associated protein-1 (KAP1) leads to increased reactivation. It was shown that in hypoxic conditions, KAP1 dissociates from the KSHV genome, which is otherwise repressed by KAP1 and other stress sensitive repressors (Zhang, Zhu et al. 2014). Overall these studies show that hypoxia activates binding of 28 REVIEW OF LITERATURE several RTA coactivators to the ORF50 promoter and induces RTA expression followed by reactivation. 5.2.7.3.2. Oxidative stress As a byproduct of cell metabolism or environmental stress, ROS can rapidly accumulate inside cells and at high enough levels their free radicals can cause severe cellular damage, which results in a stress response called the oxidative stress response. Oxidative stress has been associated with the development of many diseases such as diabetes and cancer. Cells cope with ROS by activating the NF-κB or MAPK pathways that lead to the upregulation of antioxidants (Lushchak 2014). It has been shown that hydrogen peroxide (H 2O2), a source of ROS, induces reactivation (Li, Feng et al. 2011, Ye, Zhou et al. 2011). H2O2 treatments induce all three MAPK pathways (ERK1/2, JNK and p38), which eventually activate the transcription factor c-Jun. Inhibition of MAPK pathways blocks H2O2-induced reactivation, indicating that this process is MAPK pathway-dependent (Ye, Zhou et al. 2011). Furthermore, the antioxidant N-acetyl cysteine (NAC) inhibited reactivation of PEL cells (Ye, Zhou et al. 2011). Inhibition of NF-κB increased H2O2 levels and induced lytic gene expression (Ye, Zhou et al. 2011). 5.3. Modulation of apoptosis and cell cycle in latency KSHV has evolved multiple mechanisms to escape from the host cell antiviral defense system and establish a life-long latent infection. Promoting cell division and preventing apoptosis ensures virus propagation inside the infected cells. Viral proteins expressed during latency modulate cell cycle regulators, tumor suppressors and cell death regulators and executors to elude from normal cellular stress responses and regulation (Schulz and Cesarman 2015). A good example of this is that KSHV-associated tumors infrequently carry p53 mutations and yet there is not much apoptosis detected from KS tumor sections or PEL cells (Katano, Sato et al. 2001). KSHV can disarm the function of p53 with the help of LANA, which binds both p53 and MDM2 (Sarek, Kurki et al. 2007, Chen, Hilton et al. 2010), thereby inhibiting its transcriptional activity and thus preventing p53-mediated cell cycle arrest and apoptosis (Friborg, Kong et al. 1999). Furthermore, overexpression studies have shown that LANA suppresses p53 activity, increases proliferation and induces genomic instability (Si and Robertson 2006). Additionally, LANA can manipulate the host's cell cycle and promote proliferation by interacting with cellular regulatory proteins such as pRb (Radkov, Kellam et al. 2000). 29 REVIEW OF LITERATURE Another viral protein capable of promoting cell cycle progression during latent infection is a viral homolog of human cyclin D2 (v-cyclin (ORF72)). V-cyclin shares sequence similarity with human cyclin D2 and it can form a complex with multiple CDKs, but it almost exclusively binds CDK6 and forms an active holoenzyme with it (Li, Lee et al. 1997). V-cyclin/CDK6 has broader spectrum of substrates than cellular cyclin D/CDK6 complex since it can phosphorylate substrates including Rb, histone H1, p27Kip1, p21Cip1, Bcl-2 and Nucleophosmin (NPM) (Godden-Kent, Talbot et al. 1997, Li, Lee et al. 1997, Mann, Child et al. 1999, Ojala, Yamamoto et al. 2000, Jarviluoma, Child et al. 2006, Cuomo, Knebel et al. 2008). V-cyclin/CDK6 complex is constantly active since it does not require phosphorylation of the CDK6 by CDK-activating kinase (CAK) (Child and Mann 2001, Kaldis, Ojala et al. 2001). Furthermore, it can escape from CDK inhibitor control since it is resistant against CKIs p27KIP1, p21CIP1 and p16INK4a (Swanton, Mann et al. 1997). V-cyclin/CDK6 can proceed the cell cycle from G1 phase to S-phase and enable DNA replication by phosphorylating Rb and components of DNA replication machinery (Godden-Kent, Talbot et al. 1997, Laman, Coverley et al. 2001). V-cyclin overexpression is linked to increased genomic instability which triggers p53 dependent DDR and ultimately leads to apoptosis or senescence (Verschuren, Klefstrom et al. 2002, Koopal, Furuhjelm et al. 2007). Thus v-cyclin expressing cells are able to survive only in the absence of p53 (Verschuren, Klefstrom et al. 2002, Verschuren, Hodgson et al. 2004). Interestingly it has been shown that v-cyclin/CDK6 can phosphorylate and inactivate selectively cellular Bcl-2 but not KSHV encoded viral Bcl-2 (vBcl-2, ORF16) and promote apoptosis in v-cyclin overexpressed cells (Ojala, Yamamoto et al. 2000). In addition to the inhibition of intrinsic apoptosis, KSHV is able to inhibit extrinsic apoptosis and promote cell survival. KSHV encodes a homologue of cellular FLIP (vFLIP) which contains two DED domains which bind FADD and prevent the activation of procaspase-8 by CD95 death receptor (Thome, Schneider et al. 1997). Additionally vFLIP can bind procaspase-8 directly and prevent its cleavage and activation (Belanger, Gravel et al. 2001). Furthermore, vFLIP is able to promote survival of KSHV-infected cells by activating NF-κB signaling through binding to the IκB complex (Liu, Eby et al. 2002). The maintenance of continuously active NF-Kb signaling by vFLIP is crucial for PEL cell survival (Keller, Schattner et al. 2000, Guasparri, Keller et al. 2004). 5.3.1. Manipulation of cell cycle during the lytic phase Studies so far have led to the assumption that herpesviruses induce G1 arrest to ensure efficient lytic replication (Flemington 2001). Similar findings have also been reported for KSHV lytic replication. For instance, it was shown that TPA-induced reactivation of PEL cells causes an increase in the fraction of cells in G1 phase. Similarly, overexpression studies showed that the lytic viral protein 30 REVIEW OF LITERATURE K8 (RAP) facilitated G1 arrest by activating the expression of p21 Cip-1 and by inhibiting the cyclinA/E/Cdk2 complex (Wu, Tang et al. 2002, Izumiya, Lin et al. 2003). A more recent study has identified vIRF4 as another inducer of G1 arrest. The same study showed that repressing the expression of cyclin D, a promoter of G1 progression, led to increased lytic replication (Lee, Mitra et al. 2015). Taken together, these studies suggest that K8 and vIRF4 could support lytic replication by inhibiting host cell entry into S-phase. And, as speculated by Eric Flemington in his review, it probably is the mechanism by which herpesviruses ensure a sufficient supply of nucleotides for lytic replication (Flemington 2001). 5.3.2. Manipulation of apoptosis during the lytic phase Premature cell death of the reactivated cells is a critical cellular process that the virus needs to prevent or delay in order to ensure efficient viral transcription, replication and production of infectious progeny. This is evident for KSHV from the number of viral genes in its genome devoted to the inhibition of this process. Early lytic proteins of KSHV inhibit apoptosis by interfering with the function of p53. RTA has been shown to interfere with the transcriptional activity of p53 and prevent apoptosis (Gwack, Hwang et al. 2001). K8 (RAP) has been shown to bind and inhibit p53 and apoptosis (Park, Seo et al. 2000). Overexpression studies have suggested that K8 binds to and sequesters p53 to PML bodies, thereby inhibiting apoptosis (Katano, Ogawa-Goto et al. 2001). The lytic gene ORF16 encodes a homolog of the human Bcl-2 protein called viral Bcl-2 (vBcl2). vBcl2 is anti-apoptotic protein (Sarid, Sato et al. 1997) that binds to many pro-apoptotic BH-3 proteins like Noxa, Bik, PUMA, Bak, Bax, Bid, Bim and Bad (Huang, Petros et al. 2002, Loh, Huang et al. 2005, Flanagan and Letai 2008). vBcl2 escapes from cellular regulation since it lacks the cleavage site that the human Bcl-2 protein harbors and that makes the latter sensitive to cleavage by caspase-3. In cells that are fully committed to apoptosis, this cleavage converts Bcl-2 from an anti- to pro-apoptotic protein (Bellows, Chau et al. 2000). Moreover, when complexed with CDK6, the latent viral protein v-cyclin can phosphorylate and selectively inactivate Bcl-2 but not vBcl-2 (Ojala, Yamamoto et al. 2000). Knock out of ORF16 from the KSHV genome leads to a decrease in both lytic replication and production of viral particles (Gelgor, Kalt et al. 2015, Liang, Chang et al. 2015). However, at least in cell culture systems, the anti-apoptotic function of vBcl2 was not required for lytic replication, as deletions or mutations that impaired only this function of the protein had no effect on lytic replication. Since depletion of the full protein did impair reactivation, vBcl2 must have an as of yet unidentified role in the KSHV lytic cycle (Liang, Chang et al. 2015). 31 REVIEW OF LITERATURE K7 (vIAP) is a lytic glycoprotein sharing structural similarity with the anti-apoptotic human protein survivin. vIAP inhibits apoptosis by binding active caspase-3 and blocking its function. At the molecular level, vIAP binds both activated caspase-3 and Bcl-2 to bridge these two proteins together and thus inhibits caspase-3-mediated apoptosis (Wang, Sharp et al. 2002). KSHV encodes four viral homologues of interferon regulatory factors (vIRF1-4) expressed in latent (vIRF3) and lytic infection (vIRF1,2,4) that interfere with the host antiviral responses that would otherwise lead to growth suppression and apoptosis (Moore and Chang 2003). Both vIRF1 and vIRF3 bind to p53 and impair its function (Nakamura, Li et al. 2001, Rivas, Thlick et al. 2001, Seo, Park et al. 2001, Baresova, Musilova et al. 2014). Furthermore, vIRF4 stabilizes MDM2 to degrade p53 (Lee, Toth et al. 2009) and vIRF1 interacts with ATM to inhibit the phosphorylation of p53 at Ser 15. When unphosphorylated p53 is rapidly degraded, the strong DDR that is induced during KSHV infection is impaired, and this can lead to premature cell death (Shin, Nakamura et al. 2006). A recent study that was designed to discover novel KSHV-encoded proteins that exhibit p53inhibitory function identified the late lytic structural genes ORF22, ORF25 and ORF64 (Chudasama, Konrad et al. 2015). These structural proteins inhibited p53-induced apoptosis and p53 transactivation of the apoptotic genes Bax and PIG3 (Chudasama, Konrad et al. 2015). In summary, during lytic infection, KSHV lytic proteins are able to constitutively activate cell proliferation, manipulate the regulation of the cell cycle and inhibit apoptosis in order to both prevent premature cell death and secure efficient lytic replication and production of viral progeny. 5.4. The lytic replication phase is necessary for the progression of KSHV associated malignancies Many clinical studies have shown that virus reactivation is a critical step in the appearance and progression of KS. Serological studies have revealed that detection of viral DNA in blood is associated with the appearance of new KS lesions. KSHV seropositive patients who carry KSHVinfected peripheral blood mononuclear cells (PBMC) have a 10-fold higher incidence of developing KS than patients whose PBMCs are not infected (Whitby, Howard et al. 1995, Moore, Kingsley et al. 1996, Keller, Zago et al. 2001, Cannon, Dollard et al. 2003, Engels, Biggar et al. 2003). Elevated viral loads in PBMCs correlate with the severity of KS progression (Campbell, Borok et al. 2000, Boneschi, Brambilla et al. 2001, Broccolo, Tassan Din et al. 2016). These correlations suggest that virus production is required for disease progression. Confirming this notion, treatment of KS patients with the drugs Ganciclovir or Foscarnet, both inhibitors of 32 REVIEW OF LITERATURE herpesvirus DNA synthesis, decrease the risk of KS development and slow down KS progression (Jones, Hanson et al. 1995, Martin, Kuppermann et al. 1999, Robles, Lugo et al. 1999). Interestingly although PEL cells are mostly latently infected, inhibitors of KSHV lytic replication: Ganciclovir and Cidofovir has shown to be potential therapeutic modalities in treatment of PEL (Luppi, Trovato et al. 2005, Stingaciu, Ticchioni et al. 2010, Pereira, Carvalho et al. 2014) underlining the importance of the lytic cycle on progression of the KSHV associated cancers. Lytic cycle is not only required for viral shedding and maintenance of persistent infection; it is also directly and indirectly advancing KS pathogenesis by promoting inflammation, angiogenesis and cell growth via lytic gene products (Ganem 2010, Andrei and Snoeck 2015). Therefore, inhibition of the lytic cycle or targeting of cellular processes utilized and activated by KSHV is an attractive target for therapeutic strategies. 5.5. Latest discoveries on therapeutic modalities of KSHV associated cancers Most of the current pre-clinical studies in KSHV associated cancers have been performed with inhibitors targeting cellular pathways, which are active in KSHV infected cells (Antar, El Hajj et al. 2014, Andrei and Snoeck 2015). PEL cell survival is dependent on vFLIP activated NF-κB signaling therefore several small molecule inhibitors targeting this survival pathway has shown to be potential in pre-clinical studies. As in the study where NF-κB was inhibited with BAY 11-7082 exerts a strong reduction of PEL cell survival (Keller, Schattner et al. 2000). And moreover proteasome inhibitors MG-132, lactacystin, proteasome inhibitor I and Bortezomib suppress NF-κB activity of PEL cells and promote apoptosis (Matta and Chaudhary 2005, Saji, Higashi et al. 2011). And in the recent study was discovered that by inhibition of NEDDylation with small molecule inhibitor (MLN4924) NFκB signaling was suppressed causing cell death and inhibition lytic replication of PEL cells (Hughes, Wood et al. 2015). Interestingly diallyl trisulfide (DAT), a main component of garlic oil, was shown to inhibit NF-κB and suppress PEL formation in xenograft mice (Shigemi, Furukawa et al. 2016). Immunomodulatory drugs (IMiDs) like thalidomide or second generation IMiDs lenalidomide and pomalidomide have been shown to have anti-tumor activity on multiple myeloma and other B-cell lymphomas (Shortt, Hsu et al. 2013) and recently it has been discovered that anti-tumor activity of IMiDs are based on their ability to induce degradation of B-cell specific transcription factors Ikaros family zinc finger 1 and 3 (IKZF1 and IKZF3) (Kronke, Udeshi et al. 2014). A recent study where these IMiDs were tested on PEL showed that lenalidomide and pomalidomide induced G1 arrest by 33 REVIEW OF LITERATURE downregulating IRF4 and its downstream target c-Myc. This study showed that IMiDs were targeting IKZF1 which led to downregulation of IRF4 and thus downregulation of c-Myc. And by targeting c-Myc expression with inhibitor (JQ1) targeting Bromodomain containing 4 (BRD4), antiproliferative effect of IMiDs were enhanced and co-treatment with JQ1 and lenalidomide increased survival of PEL xenograft mice (Gopalakrishnan, Matta et al. 2016). As it has been shown that c-Myc and IRF4 plays a role in the maintenance of latent infection (Li, Chen et al. 2010, Forero, McCormick et al. 2014), one would think that IMiDs would induce lytic replication however it was not detected in this study (Gopalakrishnan, Matta et al. 2016). Importantly there has been published a case report describing the successful treatment of a PEL patient with lenalidomide (Antar, El Hajj et al. 2014). And furthermore lenalidomide has shown to be potential drug against KS as case reports described successful treatment of 3 patients with HIV-related KS and partial response of advanced HIV-related visceral KS patient (Martinez, Tateo et al. 2011, Steff, Joly et al. 2013). Hepatocyte growth factor (HGF) is the ligand for the receptor tyrosine kinase c-MET and activation of this pathway modulate several downstream signaling pathways regulating cellular processes such as proliferation, survival, migration or angiogenesis. Thus HGF/c-MET pathway is promising drug candidate in anticancer therapies (Gherardi, Birchmeier et al. 2012). PEL cells have active HGF/cMET pathway (Capello, Gaidano et al. 2000) and KSHV infection is activating this pathway (Dai, Trillo-Tinoco et al. 2015). And when PEL cells were treated with c-MET inhibitor (PF-2341066) it induced G2/M cell cycle arrest and DNA damage mediated apoptosis of PEL cells and suppress tumor growth of PEL xenograft mice (Dai, Trillo-Tinoco et al. 2015). mTOR is serine/threonine kinase regulating protein synthesis in response to nutrient availability and enviromental stress (Ma and Blenis 2009). mTOR facilitate protein synthesis by phosphorylating ribosomal protein S6 kinase (S6KB1), which phosphorylate ribosomal S6 protein, which is a component of 40S ribosome (Ma and Blenis 2009). PI3K/AKT/mTOR pathway is critical for the survival of KSHV-infected cells (Sin, Roy et al. 2007, Bhatt, Bhende et al. 2010). Importantly inhibition of mTOR with Rapamycin treatment led to complete regression of classic-KS and iatrogenic KS tumors (Stallone, Schena et al. 2005, Diaz-Ley, Grillo et al. 2015). Rapamycin and its analogs were tested in KS xenograft mouse model, which was obtained by injecting subcutaneously KSHV infected endothelial cells. Rapamycin inhibited angiogenesis of xenograft tumor cells and reduced tumor growth and in KSHV infected endothelial cells secretion of VEGF-1 was inhibited (Roy, Sin et al. 2013). Rapamycin was found to have response in inhibition of cell growth of PEL cells and PEL tumor in xenograft mouse (Sin, Roy et al. 2007). Interestingly KSHV late lytic gene 34 REVIEW OF LITERATURE ORF36 encoding serine/threonine viral protein kinase (vPK) has been very recently found to resemble human S6KB1. vPK was found to phosphorylate S6 and promote protein synthesis of endothelial and epithelial cells. vPK was able to sustain S6 phosphorylation even in the presence of Rapamycin, indicating that vPK is able to phosphorylate S6 without mTORC. Furthermore vPK enhance cellular proliferation and was shown to be vital for production of viral particles. vPK was shown to induce anchorage independent growth of fibroblasts and tubule formation of HUVEC cells (Bhatt, Wong et al. 2016). Thus lytic cycle could cause resistance to Rapamycin treatment or prolonged treatment could develop drug resistance. Indeed it has been seen in PEL xenograft mice that initial response of Rapamycin treatment was turned into rapid drug resistance (Gasperini and Tosato 2009). Another example on drug resistance of lytic KSHV infected cells was seen with Nutlin-3 treatment which induces p53-mediated cell cycle arrest and apoptosis of latently infected PEL cells, but cells undergoing lytic infection were showing drug resistance against Nutlin-3 (Sarek, Kurki et al. 2007, Sarek, Ma et al. 2013). Furthermore by targeting cysteine-glutamate transporter (xCT) with smallmolecule inhibitor caspase dependent apoptosis could be induced in PEL cells and suppress tumor growth of PEL xenograft mice. However this treatment induced production of ROS and induced reactivation of KSHV (Dai, Cao et al. 2014). As indicated by these examples on current therapeutic modalities to treat KSHV induced cancers it is important not only target latently infected cells, but also inhibit lytic cycle. And as shown in a study where latent PEL cells were induced for lytic reactivation with HDAC inhibitor (SAHA) and co-treated with proteasome inhibitor (Bortezomib) which inhibited late lytic replication and release of viral progeny offers promising therapeutic modality to induce apoptosis of KSHV infected cells without increased viremia (Bhatt, Ashlock et al. 2013). 35 AIM OF THE STUDY AIMS OF THE STUDY 1. To identify the cellular targets of the KSHV encoded miRNAs (I) 2. To test if small molecule inhibitors of anti-apoptotic Bcl-2 family proteins induce cell death of KSHV infected cells and provide new anti-viral drugs (II) 3. To identify new host epigenetic regulators which are regulating KSHV lytic reactivation (III) 4. To discover protein-protein network of v-cyclin and host cell proteins 36 MATERIALS AND METHODS MATERIALS AND METHODS The materials and methods of this study are listed below, with detailed description in the original publications, here referred to by Roman numerals. Table 2. Cell lines used in this study Cell line BC-3 Description Patient derived KSHV positive human primary effusion lymohoma cell line (PEL) CZE EBV transformed Human lymphoblastoid cell line Human Burkitt lymphoma cell line Human endothelial cell line, cell hybrid of human umbilical vein endothelial cells and human lung carcinoma cell line A594 Human embryonic kidney cell line. Contain a single stably integrated FRT site at a transcriptionally active genomic locus. Tetracycline inducible SLK cells harboring recombinant KSHV (rKSHV.219, (Vieira and O'Hearn 2004)) De novo KSHVinfected E6/E7 immortalized human lymphatic endothelial cell line Adenovirus 5 immortalized primary human embryonic kidney cell line rKSHV.219-de novo infected SLK cell line DG-75 EA.hy926 HEK 293 Flp-In iSLK.219 K-iLEC QBI-HEK 293A rKSHV-SLK Source or reference National Institutes of Health AIDS Research and Reference Reagent Program (catalog # 3233 from McGrath and Ganem) Sarek, Kurki et al. (2007) Used in I, II, III ATCC I Prof. Kari Alitalo (Edgell, McDonald et al. 1983) I Invitrogen unpublished v-cyclin study Dr. J. Myoung and Prof. D. Ganem (Myoung and Ganem 2011) II, III unpublished v-cyclin study Promocell, I E6/E7 immortalization previously described in (Moses, Fish et al. 1999) QBiogene I This study 37 III MATERIALS AND METHODS SLK Originally established from a mucosal KS lesion, but later identified as human clear-cell renal-cell carcinoma cell line. rKSHV-Vero rKSHV.219 de novo infected monkey kidney cells Table 3. Primary antibodies used in this study National Institutes of II, III Health AIDS Research and Reference Reagent Program J.Vieira (Vieira and O'Hearn 2004) II, III Antibody Bap1(C-4) Source of reference Santa-Cruz Biotechnology Used in unpublished v-cyclin study Millipore MAB4603 I Santa-Cruz Biotechnology unpublished v-cyclin study Santa-Cruz Biotechnology unpublished v-cyclin study Cell signaling Asp175 I Santa-Cruz Biotechnology unpublished v-cyclin study ABI Biotechnologies III Santa Cruz Biotechnology sc-135746, (Chan, Bloomer et al. 1998) III ABI Biotechnologies III Abcam III Calbiochem III Caspase-3 Cdc2 p34 (PSTAIRE) Cdk7(C-4) Cleaved caspase-3 Histone H4(F-9) K8.1 A/B KSHV ORF57 (MTA) KSHV ORF59 LANA (HHV8ORF73) MDM2 (2A10) MDM2 (IF-2) Description Mouse monoclonal IgG antibody against human BAP1 Mouse monoclonal antibody against human caspase-3 Rabbit polyclonal IgG antibody against PSTAIRE domain of human CDK1 Mouse monoclonal IgG antibody against human CDK7 Rabbit monoclonal antibody against human cleaved caspase-3 Mouse monoclonal IgG antibody against human Histone H4 Mouse monoclonal antibody against K8.1 Mouse monoclonal antibody against ORF57 Mouse monoclonal antibody against ORF59 Rat monoclonal antibody against LANA Mouse monoclonal antibody against MDM2 Mouse monoclonal antibody against MDM2 38 III MATERIALS AND METHODS MDM2 (SMP14) Mono-HA.11(16B12) MYBBP1A p21Cip-1 p53 p53 Peg10(C-7) pH3S10 USP9Y v-cyclin γ-tubulin Mouse monoclonal antibody against MDM2 Mouse monoclonal IgG antibody against influenza hemagglutinin epitope Rabbit polyclonal IgG antibody against human MYB pinding protein 1a Rabbit polyclonal antibody against human p21Cip-1 Mouse monoclonal antibody against human p53 Rabbit polyclonal antibody against human p53 Mouse monoclonal IgG antibody against human Peg10 Rabbit polyclonal antibody against phosphorylated histone 3 serine 10 Mouse monoclonal IgG antibody against human USP9Y Rabbit polyclonal IgG antibody against vcyclin Mouse monoclonal antibody against human γ-tubulin Abcam III Covance unpublished v-cyclin study Novus Biologicals unpublished v-cyclin study Santa Cruz Biotechnology C-19, sc-397 GeneSpin DO-1 III Santa Cruz Biotechnology FL-393 Santa-Cruz Biotechnology III III unpublished v-cyclin study Upstate Biotechnology III Sigma Aldrich unpublished v-cyclin study Self-made unpublished v-cyclin study Sigma-Aldrich GTU-88 I, III, unpublished vcyclin study rKSHV.219 production and infection of SLK cells (studies II and III) The rKSHV.219 virus contains, in addition to all the KSHV genes, a gene coding for green fluorescent protein (GFP), the expression of which is under the control of a cellular constitutively active promoter (EF-1α), and a gene coding for red fluorescent protein (RFP), the expression of which is under control of the viral lytic PAN promoter (Vieira and O'Hearn 2004). rKSHV-Vero cells were used to produce infectious rKSHV.219 virus as described in (Vieira and O'Hearn 2004). Briefly, 70% to 90% confluent rKSHV-Vero cells in 100 or 150 mm dish were reactivated by adding 3- 7 ml of a recombinant baculovirus (BacK50) expressing the KSHV lytic activator ORF50 39 MATERIALS AND METHODS (RTA) for 2 hours at room temperature (RT) and 6-8 hours at 37°C at 5% CO2, which after BacK50 was removed, and replaced with complete media (DMEM:RPMI; in 40:60 ratio) containing 1,35 mM sodium butyrate (NaB, Sigma-Aldrich). Supernatants containing rKSHV.219 were collected 48 hours later and cell debris was spin down by centrifugation at 1200 rpm for 5 minutes and supernatant was filtered through 0.45 μm syringe filter (Millipore) to remove the cell debris. Virus supernatant was concentrated by ultracentrifugation at 18 000 rpm (SW-32-Ti rotor, Beckman Coulter) for 2 hours at 4°C. Virus pellet was resuspended with 100 μl PBS overnight at 4°C and concentrated virus supernatant was snap froze and stored in liquid N 2. SLK cells were plated at a density 2x105 cells per well in a six-well dish and were infected the next day with concentrated rKSHV.219 virus supernatant in the presence of 8 μg/ml polybrene to enhance their infectivity. The plates were spin-transduced by centrifugation at 1,050 g (Heraeus Multifuge) for 30 minutes RT. Cells were returned to 37°C at 5% CO 2 for 2 hours, after which the rKSHV.219 supernatant was replaced with complete DMEM media. Cells were routinely cultured in the presence of 3,5 μg/ml puromycin. Infected cells were grown in the presence of the puromycin for about two weeks to obtain a 100% infected stable cell line. Cell culture and virus reactivation (studies I, II, III and unpublished v-cyclin study) EA.hy926, SLK, rKSHV-SLK and iSLK.219 cells were cultured in a humidified 5% CO 2 atmosphere at 37°C in DMEM medium supplemented with 10% FCS, 100 U/ml penicillin G, and 100 μg/ml streptomycin. BC-3 and CZE cells were grown in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin G and 100 μg/ml streptomycin and 50 μM ß-Mercaptoethanol. HUVEC cells were grown in endothelial basal medium (Promocell) supplemented with 10% FCS, gentamicin, amphotericin and supplement kit provided with the media. To obtain KSHV miRNA expressing cells, primary and E6/E7 immortalized HUVEC cells were transduced with lentiviruses (pLENTI6/V5; Invitrogen) encoding EGFP or 10/12 KSHV miRNA cluster (K10/12) and maintained under blasticidin selection (5 μg/mL) in endothelial basal medium. To obtain immortal lymphatic endothelial cells (iLECs) and E6/E7 immortalized HUVEC cells primary human LEC and HUVEC cells (Promocell) were immortalized by the HPV oncogenes E6/E7 as previously described (Moses et al., 1999). iLEC cells were maintained in endothelial basal medium (Promocell) supplemented with 5% human AB serum (HS; Sigma, St. Louis, Mo.). To obtain K-iLEC cells, iLEC cells were infected with wild type KSHV virus as described in publication I. 40 MATERIALS AND METHODS iSLK.219 cells were grown in the presence of G418 (250 mg/ml), Hygromycin (400 μg/ml) and puromycin (10 μg/ml) and maintained as described (Myoung and Ganem 2011). To induce KSHV lytic replication, rKSHV-SLK cells were treated as described (Cheng, WeidnerGlunde et al. 2009), iSLK.219 cells were treated 0.2 μg/ml doxycycline and 1.35 mM sodium butyrate (NaB; Sigma, St Louis, MO), and BC-3 cells were treated with 1.35 mM NaB or 20 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma). HEK 293 Flp-In cell lines were cultured with high-glucose (4,5g/l) Dulbecco’s modified Eagle’s medium (HG-DMEM). All Flp-In cell lines used in this study were grown in media containing 100μg/ml Hygromycin B (Invitrogen). Induction of v-cyclin expression in Flp-In 293 cell lines was performed by addition of fresh culture medium (HG-DMEM, 50U/ml penicillin G, 50U/ml streptomycin and 2mM L-glutamine) containing 1000ng/ml tetracycline (Sigma, 5mg/ml stock in absolute ethanol) onto the cells. For immunoblotting and affinity purification cells were 70% confluent at the time of induction. miRNA inhibition with 2′O-methylated oligonucleotides and Tiny LNA treatment (study I) BC-3 cells were seeded in 6-well plates and transfected with 60 nM 2′O-methylated oligonucleotides (provided by G. Meister) which are acting as RNA analogs binding and inhibiting miRNA function. Oligonucleotides were transfected with Oligofectamine (Invitrogen) and transfections were performed according to manufacturer's instructions. Total proteins were extracted for analysis 48 h after transfection. BC-3 cells were seeded in 6-well plates and incubated with 1.5 μM short Locked Nucleic Acid oligonucleotides (tiny LNAs), which antagonize the function of miRK12-1/3/4 or the control C. elegans miR-67 by targeting miRNA seed sequence. Cells were incubated with Tiny LNAs for 48 h, or 6 days. K-iLEC cells were seeded replacing the medium twice (day 2 and 5), prior to harvesting the cells.one day before at 5×104 cells/well on 24-well plates and treated with two doses of Tiny LNA oligonucleotides (48 h+48 h) at a final concentration of 1,5 μM. Measurement of apoptosis by TUNEL assay (study I) Tiny LNA treated K-iLEC cells were induced for apoptosis with 500 μM Etoposide (Sigma Aldrich) or DMSO (mock). Cells were fixed with 4% Paraformaldehyde (EMS, Hatfield, PA) 24 h after the apoptosis induction. Apoptotic cells were detected with TdT-mediated dUTP nick end 41 MATERIALS AND METHODS labeling (TUNEL) assay according to manufacturer's instructions (In situ Cell Death Detection Kit, TMR red, Roche, Mannheim, Germany). Microscopic analysis of apoptotic cells was performed with a Zeiss Axioplan 2 fluorescent microscope (Carl Zeiss, Oberkochen, Germany). Images were acquired with a Zeiss Axiocam HRc, using Zeiss AxioVision (version 4.5 SP1) and Adobe Photoshop software (version 7.0; Adobe, San Jose, CA). Measurement of cell viability with Cell titer glo assay (study II) Efficacy and cytotoxicity testing were performed in 96-well plate format. 10 000 cells were seeded per well and cells were grown for 24 h. ABT-263 or Obatoclax (Munich, Germany) were added to the cells in 3-fold dilutions at 7 different concentrations starting from 10 μM concentration. No compounds were added to the control wells. Cell viability was measured using a Cell Titre Glo assay (CTG; Promega, Southampton, UK). Luminescence was read with PHERAstar FS plate reader (BMG Labtech, Ortenberg, Germany). Quantitative real-time PCR (study II, III and unpublished v-cyclin study) Total RNA was prepared by using the RNAeasy kit according to instructions from the manufacturer (Qiagen, Valencia, CA). RT-PCR was performed with TaqMan Reverse Transcription Reagents kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol. Real-time PCR conditions and sets of primers for ORF16 (vBcl-2) sense: 5´-GTGTTGGCCATTGAAGGGAT-3´; antisense: 5´-GTAAAACGGCCAAAGCAAAG-3´, ORF72 (v-cyclin) sense: 5´- AGCTGCGCCACGAAGCAGTCA-3´; antisense: 5´-CAGGTTCTCCCATCGACGA-3´, ORF74 (vGPCR) sense: 5’-CGCTGCACTGTTAATTGCAT-3’; antisense: 5’- GTCGCCTTAGCAGAGTGTCC-3’, ORF50, ORF57, K8.1 and human beta-actin were essentially as described previously (Dittmer, Stoddart et al. 1999, Fakhari and Dittmer 2002). For statistical evaluation of qRT-PCR data was performed as in (Cheng, Pekkonen et al. 2012). Cell spot microarray siRNA screen (study I) The custom-made siRNA library (Qiagen) was complexed with lipid transfection agents and Matrigel (BD Biosciences) and spotted/arrayed onto an untreated hydrophobic polystyrene surface. rKSHV-SLK cells were seeded onto the array as a 30 000 cells /ml suspension, and allowed to adhere for 15 min. Unattached cells were then washed off. After 48 hours, rKSHV.219 was induced for reactivation by treating cells with recombinant baculovirus (BacK50) expressing ORF 50 (RTA; a gift from J. Vieira; University of Washington) for 2 h at room temperature and 6 hours in +37°C. As a positive control for maximal reactivation, one array was in addition to BacK50 42 MATERIALS AND METHODS treatment also treated with 1.35 mM sodium butyrate (Sigma). Following induction of rKSHV reactivation, cells were incubated for an additional 30 hours, fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences) and processed for the analysis of GFP (marker of latent infection) or RFP (marker of lytic reactivation) expression using a LS400 laser scanner (Tecan) to obtain a single low resolution image of the whole array. From this image, the fluorescence of each spot was quantified with the ArrayPro Analyzer v. 4.5 (Media Cybernetics Inc) to obtain the mean intensity fluorescence value of the whole screen. Total raw mean intensities of signals for all cells per spot were normalized using pin normalization to obtain the net intensities. The net intensity value of each spot was then compared to that of the mean of the screen, used as a control, and thus calculated standard score (Z-score) was calculated for each siRNA treatment. From this analysis, siRNAs with Z-scores >2 s.d. were considered significant and cells re-imaged separately using an Olympus Scan-R high content microscope (Olympus Europe, Munich, Germany) for phenotype validation. For validation of the screen results, iSLK.219 cells were plated on a 96-well dish (Perkin Elmer) at the density of 3000 cells/well 24 hours before siRNA transfection. Cells were transfected with control or pooled siRNA oligos for MDM2 (4 siRNA oligos per gene, Qiagen) by DharmaFECT1 reagent (Dharmacon) according to the manufacturer’s instructions. 24 hours after transfection, the cells were treated with a suboptimal dose 0.05 μg/ml of doxycycline to prime the lytic induction and incubated for 52 hours. Cells were fixed with 4% PFA and the induction of RFP expression was monitored by using an automated high-content fluorescence microscope (CellInsight, Thermo Scientific). Lentiviral production and transduction (study III) Production of lentiviral supernatants was performed as described earlier (Koopal, Furuhjelm et al. 2007) with the addition of a concentration step where lentiviral supernatants were concentrated by ultracentrifugation at +4°C and with 22.000 rpm speed (SW-32-Ti rotor (Beckaman Coulter). To silence expression of MDM2 or TP53, BC-3 cells were seeded at density 5x105 /ml and transduced in the presence of 8 mg/ml polybrene (Sigma) in a 50 ml culture flask using 2 ml of concentrated frozen lentiviruses expressing two different small hairpin (shRNA) sequences specific for MDM2 (sh5-MDM2, sh3-MDM2) or p53 (sh1-p531, sh2-p53) and the corresponding scrambled controls (sh-Scr) essentially as described in (Sarek, Jarviluoma et al. 2010). To establish cell lines stably expressing sh-p53, the medium was changed at 48 hrs after transduction into a fresh medium containing 3.5 μg/ml puromycin (Sigma) and maintained thereafter. 43 MATERIALS AND METHODS Generation of the isogenic v-cyclin expressing cell line To generate the v-cyclin expressing cell lines gateway cloning technology (Invitrogen) was utilized to clone an expression plasmid (pcDNA5/FRT/TO/v-cyclin/SH/GW) containing the ORF72 (vcyclin) gene with a FRT recombination site, a hygromycin resistance gene and an N-terminal Streptag and a Hemagglutinin epitope (SH)-tag. The expression of this construct is under the control of tetracycline (tet)-operated (TO) CMV promoter. Two independent clones of v-cyclin expression plasmids were cloned and named v-cyclin 1 (vcyc1) and v-cyclin 2 (vcyc2) and a control plasmid expressing only HA- and Strep-tags (Ctrl). pcDNA5/FRT/TO/v-cyclin (1 and 2)/SH/GW expression plasmids and pOG44 plasmid expressing Flippase (Flp) recombinase enzyme were co-transfected into HEK293 Flp-In cells (Invitrogen). Co-transfection induced Flp mediated homologous recombination between FRT sites of the cellular genome and expression plasmid. Hygromycin resistance gene enables antibiotic selection of the isogenic v-cyclin cell clones. Affinity purification and silver staining (unpublished v-cyclin study) To determine binding partners of v-cyclin, affinity purification was performed by utilizing the Strep-tag® (IBA) in the N-terminus of the v-cyclin protein. The expression of the protein was induced for 24h by tetracycline (1000ng/ml) of the following 293 HEK Flp-In cell lines: v-cyclin1, v-cyclin2 and empty, cultured in six 135 cm2 culture flasks. Thereafter the cells were washed once with 1x PBS, detached with Trypsin- ethylenediaminetetraacetic acid (EDTA), pooled together and harvested by centrifugation with 153 x g for 5 minutes. Cell pellets used in this study were all snap-frozen with liquid NO2 and thawed on ice in the presence on lysis buffer HNN (50mM HEPES, pH 8; 150mM NaCl; 50mM NaF; 1mM PMSF; 1,5mM Na-vanadate; 0,5% NP-40; 1x protease inhibitor cocktail (cOmplete, Roche)) on the day of purification. Thawed pellets were homogenized into lysis buffer with Dounce homogenizator, subjected to rotary-incubation for 10 minutes and centrifuged 17 000 x g for 30 minutes at 4°C to separate soluble proteins from cell debris. Individual supernatants were filtered through Poly Prep chromatography columns (Bio-Rad) to further purify remaining lipids and debris from samples and applied further onto Strep-Tactin columns (IBA) equilibrated with wash buffer (100mM Tris-HCl pH 8.0, 150mM NaCl, 1mM EDTA) and HNN lysis buffer respectively. Unbound proteins were washed from the column first twice with HNN lysis buffer followed by 5 times with plain HN buffer (50mM HEPES, pH 8; 150mM NaCl) without protease inhibitors or NP-40. Elution was performed with HN-buffer containing 1mM biotin to outcompete bound strep-tag. Eluates were divided into two aliquots and subjected to analysis by mass spectrometry or immunoblotting to identify proteins pulled-down with the v-cyclin. 44 MATERIALS AND METHODS To monitor purification of v-cyclin bait protein along the process, small aliquots were taken after clarification of the lysate, from the first flow-through, and from final eluate. These samples were then applied into two 15% polyacrylamide gels and subjected to silver staining and western blot against anti-HA antibody to verify enrichment of the bait and its main binding partner respectively. Silver staining was performed according to PageSilver Silver Staining Kit (Fermentas) instructions. SDS-PAGE and Western Blotting (unpublished v-cyclin study) Each 293 Flp-In cell line was washed once with 1x phosphate buffered saline (PBS), detached with Trypsin- EDTA (1:250 Trypsin BD Difco) and harvested by centrifugation at 153 x g for 5 minutes. Cell pellets were lysed into ELB lysis buffer 150mM NaCl; 50mM N-2-hydroxyethylpiperazine-N2-ethanesulfonic acid (HEPES), pH 7.4; 5mM EDTA; 0.1% Igepal; 1mM dithiothreitol DTT and 1x cOmplete protease inhibitor cocktail (Roche, dissolved into the ELB) vortexed shortly and passed through a 0,6mm needle (Microlance 3) to enhance lysis. Lysates were clarified by centrifugation at 17 000 x g for 20 minutes at 4°C. Protein concentration was measured from the supernatant with Bio-Rad protein assay reagent. Protein standards were constituted from 10mg/ml bovine serum albumin (BSA) stock (New England Biolabs). For sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 5x Laemmli sample buffer (1M Tris-Cl, pH 6.8; 5mM EDTA; 7,5% SDS; 50% glycerol; 5% β-mercaptoethanol; 0.02% bromophenol blue) was applied to each sample to a final concentration of 1x followed by boiling of the samples for 5 minutes at 95°C. Proteins from the samples were separated together with PageRuler Plus prestained protein ladder (Thermo Scientific) on 8-,12- or 15% sodium dodecyl sulphate-polyacrylamide gels. After the SDS-PAGE, proteins were transferred to nitrocellulose membrane (Protran, Whatmann) at room temperature for 45- to 1 h 45 minutes with semi-dry transfer using Owl electroblotting system (Thermo Scientific) in semi-dry blotting buffer (47,8mM Tris; 38,6mM Glycine; 0,037% SDS; 20% methanol). The non-specific binding sites in the membrane were blocked with 5%(w/v) skim milk (in 1x Trisbuffered saline (TBS), 0,05% Tween20; 0,02% Sodium azide) (TBST) for 30 minutes at room temperature followed by overnight incubation with the primary antibody at +4°C. Analysis of v-cyclin interacting proteins with LC-MS/MS (unpublished v-cyclin study) In order to identify v-cyclin interacting proteins from the affinity purified samples, the proteins were reduced with 5 mM tris (2-carboxyethyl)phosphine (TCEP), alkylated with 10mM iodacetamide, and digested overnight to peptides with 1μg trypsin (Promega). After the digestion peptides were subjected to reverse-phase liquid chromatography and the eluting peptides analyzed using Orbitrap Elite ETD (Thermo Scientific) mass spectrometer (Institute of Biotechnology 45 MATERIALS AND METHODS Proteomics Unit) by our collaborator Docent Markku Varjosalo. The resulting LC-MS/MS spectra from the analysis were searched using SEQUEST spectrometry data analysis program after which the data was subjected to a stringent contaminant filter, a database compiled from three hundred individual affinity purification pull down experiments. All the proteins that appeared in over 10% of the database’s pull down experiments and had a match in the obtained SEQUEST data, were considered contaminants and discarded when compiling the final list of v-cyclin interacting proteins. In addition, the numerical values of common interacting peptides from HEK293 empty sample were subtracted from the vcyc1- and vcyc2 samples to obtain number of interactions resulting from the presence of v-cyclin solely. 46 RESULTS AND DISCUSSION RESULTS AND DISCUSSION 1. KSHV-encoded miRNAs target effector caspase 3 to inhibit apoptosis in latently infected cells (I) 1.1 KSHV-encoded miRNAs target caspase 3 KSHV-encoded miRNAs deregulate host cell proliferation, apoptosis and cell survival and have an important role in the development of KSHV-associated cancers (Zhu, Haecker et al. 2013). It is thus important to investigate the function of these virally encoded miRNAs and identify their cellular targets. To this end, we established two cell lines, DG-75 (derived from B-cells) and EA.hy926 (derived from endothelial cells), stably expressing the intronic cluster of KSHV-encoded miRNAs (miR-K12-1 to miRK12-9 and miR-K12-11 named K10/12), and performed microarray analysis of cell lines stably expressing the cluster of KSHV encoded miRNAs. As a control we used EGFP expressing DG-75 and EA.hy926 cells. B-cells and endothelial cells were chosen because they represent the model system for KSHV-associated diseases (Ganem 2010). Global gene expression was analyzed from these cell lines by Affymetrix Human Genome U133 Plus 2.0 microarrays. Transcripts with at least one matching seed sequence to one of the KSHV miRNA and whose expression was at least 1.4 fold lower than in the EGFP control cells were considered as putative KSHV miRNA target. The results of this analysis revealed hundreds of putative KSHV miRNA targets (Putative KSHV miRNA targets in DG-75 cells are listed in dataset S2 and in EA.hy926 cells are listed in dataset S3 in I). The findings from the microarray study were validated with a reporter gene assay in 293A cells cotransfected with plasmids encoding the KSHV miRNA cluster (pCDNA-K10/12) and the firefly luciferase gene fused with the 3’UTR of the putative miRNA target mRNA (psi-CHECK-2). We decided to validate 16 putative targets related to cell cycle, apoptosis and DNA damage. As a positive control in this assay we used the previously validated KSHV miRNA target osteopontin SPP1 (Samols, Skalsky et al. 2007). When compared to the control EGFP-expressing cells, a significant downregulation of the luciferase signal (30-50%) was observed when the K10/12 cluster was co-expressed with the 3’ UTR of the SPP1 positive control and the newly identified targets RAD51AP1 (RAD51-associated protein) and CASP3 (Fig. 4B in I). The CASP3 gene codes for the apoptosis effector caspase 3 (Hengartner 2000). To confirm that the KSHV miRNAs target the expression of endogenous caspase 3, we established primary and E6/E7 immortalized endothelial cell lines (HUVEC) stably expressing the K10/12 miRNA cluster or EGFP as a control. The endogenous protein levels of caspase 3 were analyzed by immunoblotting. 47 RESULTS AND DISCUSSION When compared to the EGFP expressing control HUVEC, a clear reduction of the endogenous caspase 3 protein levels was observed in the K10/12 expressing HUVEC (Fig. 7D in I), thus confirming the results of the transcriptomic analysis. Another putative KSHV miRNA target validated from microarray study was RAD51AP1, which is involved in the repair of DSBs in homologous recombination (HR) (Dunlop, Dray et al. 2012). KSHV infection has been reported to induce chromosomal instability of primary endothelial cells and genomic instability is also observed in PEL cells (Pan, Zhou et al. 2004, Nair, Pan et al. 2006). Moreover, KSHV infection induces DDR (Verschuren, Hodgson et al. 2004, Koopal, Furuhjelm et al. 2007) and the activation of ATM is required for the maintenance of latency (Jha, Upadhyay et al. 2013, Singh, Dutta et al. 2014). How the downregulation RAD51AP1 contributes to viral infection, genome instability and DDR remains to be established. Our transcriptomic analysis showed that the mRNA level of CASP3 and endogenous protein levels of caspase-3 were significantly decreased upon expression of the KSHV miRNA cluster. Interestingly similar results have been obtained with EBV encoded miRNAs as they have been found to target CASP3 directly and indirectly by suppressing BAD and to inhibit apoptosis to maintain latent infection (Vereide, Seto et al. 2014, Lin, Tsai et al. 2015, Kim, Choi et al. 2016). Next we validated is KSHV K10/12 miRNA cluster inducing downregulation of CASP3 in KSHV infected cells. 1.2 Depletion of KSHV miRNAs upregulates caspase 3 expression in KSHV-infected PEL cells As we were able to demonstrate that cells expressing KSHV miRNAs have reduced levels of endogenous caspase 3, we studied whether the downregulation of caspase 3 also occurs in KSHVinfected cells and if this effect is due to the expression of the KSHV miRNAs. To address these questions, we used BC-3 cells, a KSHV-naturally-infected cell line derived from a patient affected by primary effusion lymphoma (PEL). The expression of KSHV miRNAs identified to target CASP3 (Fig 5B in I, miR-K12-1, K12-3, K12-4-3p) were silenced with 2’-O-methylated (2’OMe) antisense oligoribonucleotides (Meister, Landthaler et al. 2004) or short Locked Nucleic Acid oligonucleotides (tiny LNAs; (Obad, dos Santos et al. 2011)). As a control, cells were treated with antisense oligonucleotides against a non-relevant miRNA (miR-67). Compared to the control treated cells, we could detect a modest but significant upregulation of caspase 3 mRNA and protein levels in BC-3 cells treated with antisense oligos against the viral miRNAs (Fig. 7E-G in I). This result indicates that KSHV-encoded miR-K12-1, miR-K12-3 and miR-K12-4-3p target the caspase 3 expression in PEL cells. Apoptosis is not frequently detected in KS or PEL tumors 48 RESULTS AND DISCUSSION (Katano, Sato et al. 2001). And importantly in agreement with our results, expression of cleaved (active) caspase 3 is rarely observed in KS tumors (Ramos da Silva, Bacchi et al. 2007). CASP3 genetic polymorphism is involved in pathogenesis of cancer (Yan, Li et al. 2013) and CASP3 downregulation is associating with chemoresistance, metastasis and poor prognosis in breast cancer and non small cell lung carcinoma (Volm, Mattern et al. 1999, Devarajan, Sahin et al. 2002, Okouoyo, Herzer et al. 2004). Interestingly cellular miRNAs miR-137 and miR-224 have been identified to inhibit CASP3 in lung carcinoma cells and miR-137 is inducing cisplatin resistance of lung adenocarcinoma (Cui, Kim et al. 2015, Su, Ku et al. 2016). Therefore CASP3 inhibition by KSHV miRNAs miR-K12-1, miR-K12-3 and miR-K12-4-3p could cause chemoresistance of PEL and KS patients. Thus we wanted to investigate whether the miRNAs that target caspase 3 expression would contribute to the survival of infected cells by inhibiting apoptosis and providing protection against the cellular anti-viral response, which usually leads to cell death. 1.3 KSHV miRNAs inhibit apoptosis in KSHV-infected endothelial cells The effect of KSHV miRNAs on the induction of apoptosis was studied in a KSHV-infected lymphatic endothelial cell line (K-iLEC) model. The KSHV miRNAs targeting caspase 3 were silenced as before and apoptosis was induced chemically by treating the cells with etoposide, a DNA damage and apoptosis-inducing small molecule inhibitor of cellular topoisomerases. The number of apoptotic cells was determined by immunofluorescence staining of cleaved caspase 3 and by measuring the levels of nicked DNA (a marker of apoptosis) using the TUNEL assay (Fig 8A-B in I). Quantitative analysis after fluorescence imaging revealed 2.3 fold more apoptotic cells in LNA-miR-K12-1/3/4 treated samples (Fig 8C in I) indicating that there is more apoptosis in KSHV-infected endothelial cells when miR-K12-1, K12-3 and K12-4-3p are silenced. These miRNAs can therefore effectively inhibit apoptosis in KSHV-infected endothelial cells. In summary, this study shows that KSHV miR-K12-1, K12-3 and K12-4-3p inhibit expression of caspase 3 in B-cells, endothelial cells and in the KSHV-infected PEL cells. Moreover, these miRNAs inhibit apoptosis of latently infected endothelial cells challenged with etoposide. KSHV has evolved multiple mechanisms to inhibit apoptosis and, as shown in this study, the virally encoded miRNAs add another 'weapon' to the viral arsenal against host cell anti-viral mechanisms. In addition to the miRNA-silencing of CASP3 described here, other studies have shown that KSHV has other means to inhibit the function of caspases. It has recently been shown that when KSHVinfected cells were artificially induced to undergo apoptosis, the viral latent protein LANA - which is very abundant in KSHV-infected cells - was found to be cleaved by caspase 1 and 3. It was 49 RESULTS AND DISCUSSION proposed that LANA can impair apoptosis by acting as a caspase 'decoy' substrate (Davis, Naiman et al. 2015). Another interesting finding suggests that caspase 3-activated apoptosis could induce reactivation of KSHV and other herpesviruses (Prasad, Remick et al. 2013). This would suggest that herpesviruses are able to sense the induction of caspase 3-dependent apoptosis and resume lytic replication and virion release when apoptosis cannot be inhibited or delayed adequately. Hence, the inhibition of caspase 3 could serve as an additional mechanism to maintain latent infection. Consistent with this hypothesis, K12-3 and K12-11 miRNAs were shown to inhibit lytic gene expression by targeting transcription factors required for the transcription of ORF50, the initiator of the lytic cascade (Plaisance-Bonstaff, Choi et al. 2014). Multiple KSHV miRNAs have been shown to target regulators of apoptosis. Tandem array-based expression screen analysis of KSHV miRNA expressing B-cells and KSHV-infected endothelial cells revealed that miR-K6, miR-K9 and mir-K10a/b target Bcl-2-associated factor (BCLAF1) (Ziegelbauer, Sullivan et al. 2009). Moreover, miR-K11 targets SMAD5 and miR-K11 targets TGFβ type II receptor to inhibit TGFβ signaling and promote cell survival (Lei, Zhu et al. 2012, Liu, Sun et al. 2012). Recently it has been discovered that KSHV can transform the primary rat mesenchymal precursor cell line (MM), which has provided an important tool to study mechanisms of KSHV-induced tumorigenesis (Jones, Ye et al. 2012). These cells were used to study the role of the KSHV miRNAs in tumorigenesis. In this study, a cluster of 10 viral miRNAs were genetically removed from the KSHV genome and MM cells were infected with this mutant virus. Importantly, infection did not induce transformation, but instead induced cell cycle arrest or apoptosis (Moody, Zhu et al. 2013). This study demonstrates how KSHV miRNAs play a major role in the oncogenesis of KSHV-induced cancers by deregulating host cell proliferation, apoptosis and cell survival. 2. Targeting cellular anti-apoptotic factors to specifically kill virus-infected cells (II, unpublished data) 2.1 KSHV-infected cells are resistant to the Bcl-2 inhibitor ABT-263 The cellular anti-apoptotic proteins Bcl-2 and Bcl-xL are overexpressed in many cancers, which is related to poor prognosis and resistance to chemotherapy treatment (Tsujimoto, Finger et al. 1984, Raffo, Perlman et al. 1995, Friess, Lu et al. 1998). The same factors are also overexpressed in KS, and expression of Bcl-2 increases during disease progression (Foreman, Wrone-Smith et al. 1996, Morris, Gendelman et al. 1996). Hence, targeting cellular anti-apoptotic pathways could be a way to specifically kill virus-infected cells, thus providing a new antiviral strategy. 50 RESULTS AND DISCUSSION As a proof of principle, we tested a small molecule inhibitor, ABT-263, which is a BH-3 mimetic drug that binds and inhibits the anti-apoptotic functional portions of Bcl-2, Bcl-xL and Bcl-w. This inhibition resulted in the induction of apoptosis of cancer cells (Wong 2011, Moldoveanu, Follis et al. 2014). Cells infected with several different viruses, including KSHV, were treated with different concentrations of ABT-263 and cell viability was measured by luciferase-based Cell titer glo assay (CTG, Promega). The results of this analysis showed that ABT-263 could accelerate cell death of various cell types infected with influenza A and B, Bunyamwera, Semliki Forest, Sindbis, herpes simplex 1 and 2, vaccinia and measles (Fig. 2C-D in II). However, the inhibitor did not kill iSLK.219 cells, a cell line latently infected with a recombinant KSHV ((Myoung and Ganem 2011); Fig. 2D in II, Figure 6A, no ind)). To study the effect of ABT-263 on de novo infected cells, we infected SLK cells (the non-infected parental cell line of iSLK.219 cells) with recombinant KSHV (rKSHV.219, (Vieira and O'Hearn 2004)) and simultaneously with the virus we added the inhibitor to the cells in 3-fold dilutions at 7 different concentrations starting from 10 μM. Cell viability was measured by the CTG assay 24 hours post infection. The dose response curve showed no difference in the cell viability between non-infected (Mock) and rKSHV.219-infected cells (Figure 5). Figure 5. ABT-263 inhibitor did not induce cell death of de novo infected SLK cells. Mock or rKSHV.219-infected SLK cells were treated with indicated concentrations of ABT-263 drug and 24 hours post infection cell viability was measured to determine the dose response curve. These results demonstrate that in contrast to the other viruses tested in this study, KSHV-infected cells do not respond to the Bcl-2 inhibitor, ABT-263. Probably indicating that survival of latent KSHV infected cells and cell survival upon KSHV infection is not dependent on Bcl-2, Bcl-xL or 51 RESULTS AND DISCUSSION Bcl-w proteins. This could be explained by the viral inhibition of caspase 3 and caspase-8 and thus apoptosis during latent infection (Figure 8 A-C in I)(Belanger, Gravel et al. 2001, Davis, Naiman et al. 2015). Another mechanism behind the ABT-263 resistance of the latent KSHV infected cells could be the viral inhibition of p53 (Rivas, Thlick et al. 2001, Sarek, Kurki et al. 2007, Chen, Hilton et al. 2010, Baresova, Musilova et al. 2014), since it has been shown that Bcl-xL is binding and inhibiting p53 (Follis, Chipuk et al. 2013), therefore inhibition of Bcl-xL by ABT-263 could lead release of p53 and induction of apoptosis, which is probably inhibited by KSHV latent proteins. 2.2 ABT-263 did not induce cell death of the lytic KSHV-infected cells To test whether ABT-263 treatment would cause cell death of cells undergoing active KSHV lytic replication, we induced the lytic cycle in iSLK.219 cells with doxycycline and NaB. 24 hour post induction (hpi) we added the drug as in the earlier experiments followed by monitoring of cell viability with the CTG assay. ABT-263 did not cause significant cell death during the latent or lytic phase of KSHV life cycle in iSLK.219 cells (Fig. 6A). The inhibitor was also tested in BC-3 PEL cells. To induce the lytic cycle of the virus, the cells were treated with TPA 24 hours prior ABT-263 treatment, and cell viability was measured 24 hours later with the CTG assay. As shown in Figure 4B, ABT-263 did not cause cell death of either latent or lytic BC-3 cells. Figure 6. Dose response curve of non-induced (latent KSHV) and induced (lytic viral cycle) iSLK.219 cells (A) and DMSO (-TPA) or TPA-induced BC-3 cells (B). In summary, our data demonstrate that lytic KSHV-infected cells were resistant to the ABT-263 drug, indicating that the viability of KSHV-infected cells is not dependent on the cellular Bcl-2, Bcl-xL or Bcl-w proteins. Probably the function of anti-apoptotic Bcl-2 proteins is redundant in lytic KSHV infected cells as KSHV encoded vIRF1 is able to bind and inhibit Bim and Bid during lytic infection (Choi and Nicholas 2010, Choi, Sandford et al. 2012) and vIAP is inhibiting caspase52 RESULTS AND DISCUSSION 3 (Wang, Sharp et al. 2002). And furthermore multiple lytic proteins inhibiting p53 mediated apoptosis (Gwack, Hwang et al. 2001, Katano, Ogawa-Goto et al. 2001, Nakamura, Li et al. 2001, Seo, Park et al. 2001, Chudasama, Konrad et al. 2015). The most promising viral candidate behind the ABT-263 resistance of lytic KSHV infected cells is the viral homolog of Bcl-2 protein: ORF16 (vBcl-2) binding multiple pro-apoptotic BH-3 proteins (Sarid, Sato et al. 1997, Huang, Petros et al. 2002, Loh, Huang et al. 2005, Flanagan and Letai 2008). To investigate this possibility further, we monitored the kinetics of expression of the ORF16 gene at different stages of the lytic cycle that was induced in iSLK.219 or BC-3 cells by the addition of doxycycline/NaB or TPA, respectively. RNA samples were collected at 0, 4, 8, 12, 24, 30 and 48 hours post induction (hpi) and the expression of ORF16 was analyzed by qRT-PCR. This analysis revealed a fast and robust upregulation of ORF16 expression in iSLK.219 cells at 4 h after reactivation, followed by a linear increase until 30 h (Fig. 7A). In BC-3 cells, the expression of ORF16 was detectable at 4 hpi and it increased linearly during the time course of TPA treatment (Fig. 7B). Figure 7. Expression of ORF16 (vBcl-2) in the KSHV-infected lytic-induced cells. A) qRT-PCR analysis of iSLK.219 cells induced for lytic cycle with doxycycline and NaB treatment prior to RNA extraction at indicated time points. B) qRT-PCR analysis of BC-3 cells induced with TPA treatment at indicated time points. These data indicate that KSHV-infected cells efficiently express the product of the ORF16 gene both in the early and late phases of the lytic cycle. Expression of ORF16 coexists with the ABT-263 treatment as it was administered to cells 24 hours post reactivation thus it could explain the resistance of lytic iSLK.219 and BC-3 cells on ABT-263 treatment (Figure 6). Interestingly, a recently published study of vBcl-2 protein structure revealed that the BH3 binding groove of the viral protein is structurally and functionally similar to another cellular anti-apoptotic protein, Mcl-1 (Foight and Keating 2015). Moreover expression of Mcl-1 have been identified to be mediating resistance of breast cancer, NHL and multiple myeloma cells to Bcl-2/Bcl-xL/Bcl-w inhibitors 53 RESULTS AND DISCUSSION (Phillips, Xiao et al. 2015, Xiao, Nimmer et al. 2015, Punnoose, Leverson et al. 2016). Resistance of lytic KSHV infected cells to ABT-263 treatment could be explained by anti-apoptotic function of vBcl-2 and its resemblance with Mcl-1 and the possibility that ABT-263 could not bind vBcl-2 and inhibit its function. Accordingly, we next tested if inhibitor of Mcl-1, Obatoclax, could induce cell death of KSHV infected cells. 2.3 Obatoclax induces cell death in the lytic KSHV-infected cells To test whether Obatoclax could cause cell death of the latent and lytic KSHV-infected cells, the lytic cycle in iSLK.219 cells were induced with doxycycline and NaB. At 24 hpi cells were treated with the same concentrations as in Figures 5 and 6 and cell viability was monitored with the CTG assay. Obatoclax treatment did not induce a remarkable amount of cell death in the latent iSLK.219 cells (Figure 8, no ind.). As for the lytic iSLK.219 cells, Obatoclax treatment induced significant cell death at the lower concentrations of 123-370 nM (Figure 8, ind.). Figure 8. Obatoclax induces cell death in lytic KSHV-infected cells. iSLK.219 cells were induced for lytic cycle with doxycycline and NaB and 24 hpi cells were treated with indicated doses of Obatoclax and cell viability was analyzed with CTG assay. No in= cells treated with DMSO prior Obatoclax; ind=cells treated with doxycycline and NaB prior Obatoclax. These results indicate that Obatoclax is able to specifically target and kill the lytic KSHV-infected cells. Importantly, a recent study has identified that Obatoclax is able to also kill latently infected PEL cells but that this effect requires both higher concentrations and a longer treatment time of 4872 hrs (Nayar, Lu et al. 2013), indicating that cells infected with lytic virus are more sensitive to the Obatoclax treatment. These findings indicate that the survival of KSHV-infected cells may be 54 RESULTS AND DISCUSSION dependent on the Mcl-1 protein. This is further supported by a recent study in which the dependency of KSHV-infected cells on the individual anti-apoptotic Bcl-2 proteins was screened with a BH3 profiling method which revealed that survival of latent PEL cells is indeed dependent on Mcl-1 and vBcl-2 (Cojohari, Burrer et al. 2015). Interestingly this study demonstrated that the ABT-737 inhibitor targeting Bcl-2 and Bcl-XL or the MIM1 inhibitor specifically targeting only Mcl-1 could not kill the latently infected PEL cells (Cojohari, Burrer et al. 2015). Hence it appears that KSHV simultaneously utilizes both Mcl-1 and vBcl-2 to ensure its survival. Therefore the pan Bcl-2 inhibitor like Obatoclax inhibiting in addition to Mcl-1 also Bcl-2, Bcl-xL and Bcl-w, could offer a new anti-viral drug candidate to treat KSHV-associated cancers. To study potential anti-viral effect of Obatoclax further, it is important to address can Obatoclax inhibit reactivation and release of viral progeny. Also detailed mechanism of Mcl-1 in lytic cycle of KSHV infection should be addressed further. Results with IAV mouse model indicated that when treated with ABT-263 drug mice had significantly lower survival rate than mock treated animals (Figure 7 A in II) and furthermore ABT263 induced imbalanced cytokine production of IAV infected mice (7 E-F in II). Causing potential thread to ABT-263 treated cancer patients with underlying or apparent viral infection. KSHV infection is associated with KICS which has similar symptoms as seen in ABT-263 treated IAV mice; as these patients have elevated cytokine levels and higher viral loads (Uldrick, Wang et al. 2010, Polizzotto, Uldrick et al. 2012), thus it is important to keep in mind that treatment of patients with KSHV associated cancer could have a higher risk to develop KICS. Therefore it is important to test can Obatoclax and other Bcl-2 inhibitors induce lytic cycle of KSHV infected cells and further test these inhibitors in animal models like PEL xenograft mice. The research on the host cell mechanisms utilized by tumor viruses enables to discover potential new anti-viral drugs towards KSHV and its associated diseases. The KSHV lytic cycle has an instrumental role in the emergence and progression of KS (Whitby, Howard et al. 1995, Moore, Kingsley et al. 1996, Keller, Zago et al. 2001, Cannon, Dollard et al. 2003, Engels, Biggar et al. 2003). Therefore it is highly important to study the cellular mechanisms that are necessary for the viral lytic program. 55 RESULTS AND DISCUSSION 3. KSHV utilizes the p53 stress response to induce a G2/M cell cycle checkpoint for efficient lytic replication (III) 3.1 siRNA screen identifies MDM2 as a novel regulator of KSHV reactivation To discover new host cell regulators of KSHV reactivation, we performed an RNAi screen using a custom-made library of siRNA oligos targeting 615 different human epigenetic factors and their regulators. In this screen, we used an SLK cell line (Herndier, Werner et al. 1994, Sturzl, Gaus et al. 2013) infected with a recombinant reporter KSHV (rKSHV.219) (Vieira and O'Hearn 2004) as a model system for reactivation. The rKSHV.219 virus contains, in addition to all the KSHV genes, a gene coding for green fluorescent protein (GFP), the expression of which is under the control of a cellular constitutively active promoter (EF-1α), and a gene coding for red fluorescent protein (RFP), the expression of which is under control of the viral lytic PAN promoter (Vieira and O'Hearn 2004). With this reporter virus the latently infected cells can be visualized by expression of GFP and the lytic replication-induced cells can be monitored by the expression of RFP. The screen was done by using a cell-spot microarray technique as detailed in the materials methods section (CSMA; (Rantala, Makela et al. 2011). A schematic of the siRNA screen workflow is depicted in Fig. S1A of publication III. The screen identified 20 novel cellular factors that increased the efficiency of KSHV reactivation when depleted. One of the strongest hits was MDM2, an ubiquitin E3 ligase and master regulator of p53 protein stability (Fig. S1 B in III). Using additional, different siRNA oligos than those used in the screen to target MDM2, this hit was validated in the rKSHV.219-infected SLK cell clone (iSLK.219), in which KSHV reactivation can be induced by doxycycline treatment (Myoung and Ganem 2011). These validation experiments revealed that the silencing of MDM2 led to a 3-fold higher RFP expression in iSLK.219 cells, indicating increased reactivation (Fig. 1 A-B in III). 3.2 Knockdown of MDM2 enhances KSHV reactivation To validate the finding from the siRNA screen using a biologically more relevant cell line, we depleted MDM2 mRNA expression in PEL cells (BC-3) by using lentivirus-mediated short hairpin RNA (shRNA) treatments (sh-MDM2 and control shRNA; sh-Scr). To determine the effect of MDM2 depletion on spontaneous reactivation we measured the relative amount of lytic transcripts by qPCR. This analysis revealed that MDM2 depletion led to increased mRNA levels of the lytic transcripts expressed in the immediate early- (ORF50), delayed early- (ORF57 and vGPCR) and late- (K8.1) lytic replication phases (Fig. 1C in III). To examine the effect of MDM2 silencing on maximal reactivation, we induced reactivation with TPA treatment and measured lytic gene expression at different phases of the reactivation cascade (4, 8 and 48h). Importantly, MDM2 56 RESULTS AND DISCUSSION depletion enhanced expression of all the measured lytic genes after 4 and 8 hours of TPA induction (Fig 1D in III), while at the later time point (48 h) only the expression of K8.1 was significantly increased (Fig 1D in III). Altogether these results demonstrate that knockdown of MDM2 enhances the expression of lytic genes in both non-induced and TPA-induced PEL cells, thus supporting that MDM2 regulates both the spontaneous and chemically-induced KSHV reactivation. Importantly, similar results have been obtained with adenovirus, another DNA virus, by showing that knockout of MDM2 and MDM4 leads to increased viral gene expression (Yang, Zheng et al. 2012). Interestingly, previous results also indicate that the MDM2 antagonist Nutlin-3 induces lytic reactivation of KSHV-infected endothelial cells, strengthening our hypothesis that MDM2 is a negative regulator of KSHV reactivation (Ye, Lattif et al. 2012). MDM2 is highly expressed in the PEL tumors in comparison to other B-cell lymphomas (Petre, Sin et al. 2007, Sarek, Kurki et al. 2007). Moreover, our group and others have demonstrated that during the latency phase, LANA forms a ternary complex with p53 and MDM2 and that this complex is disrupted by Nutlin-3 treatment (Sarek, Kurki et al. 2007, Chen, Hilton et al. 2010). These findings therefore suggest that MDM2 could have an important role in the maintenance of latent infection, probably by sequestration of p53 together with LANA. Our earlier studies indicate that Nutlin-3 treatment induces p53-mediated apoptosis of latently infected PEL cells but, interestingly, cells undergoing lytic infection were refractory to the cell killing effect of Nutlin-3 (Sarek, Kurki et al. 2007, Sarek, Ma et al. 2013). This implies that the restored p53 somehow prevents the cells from undergoing apoptosis and instead favors the cells infected with the lytic KSHV. Therefore, we wanted to examine the role of this tumor suppressor in KSHV reactivation in more detail. 3.3 Viral reactivation leads to a p53 response in PEL cells To comprehensively study the role of p53 in KSHV reactivation, we analyzed the occupancy of p53 on the infected host cell chromatin and viral genomes with ChIP sequencing (ChIP-seq) analysis of reactivation-induced, TPA-treated BC-3 cells. In this assay, Nutlin-3-treated cells were used as a positive control. Analysis of the input samples showed p53 stabilization at 24 h upon TPA treatment and a strong induction of p53 in the positive control sample (Fig. 2D in III). To our surprise, the ChIP-seq analysis showed no p53 binding on the KSHV genome, indicating that p53 is not functioning through binding to the viral genome but rather is activated by the virus and regulates the host cell gene expression (Fig. 2 B in III). Analysis of p53 binding sites on the host cell genome demonstrated that a partial p53 response occurred upon TPA-induced reactivation (Fig. 57 RESULTS AND DISCUSSION S3A in III). Gene Ontology (GO) analysis of these p53 binding sites revealed genes related to cellular processes such as p53 regulation (MDM2), cell cycle arrest (CDKN1A), DNA repair (p53R2) and apoptosis (PAG608) (Fig. 2A and S3C in III). Next, we decided to focus on the cell cycle arrest function of the p53 response and validate the induction of CDKN1A gene expression by immunoblotting its gene product, p21Cip-1, from the 24h TPA and 8h Nutlin-3-treated BC-3 cells (Lane 2 in Fig. 2D in III). Importantly, we could detect an equally strong p21 Cip-1response in both TPA and Nutlin-3 treated cells (Lane 2 in Fig. 2D in III). Moreover, we found a potent p21 Cip-1 response in both the NaB-induced BC-3 and TPA/Dox or NaB/Dox-induced iSLK.219 cells (Fig. 2E in III), indicating that the p21Cip-1 response is not limited to PEL cells but also occurs in other infection models upon KSHV reactivation. In summary, our results demonstrate that KSHV reactivation induces both a p53 response and p21Cip-1 upregulation in PEL and iSLK.219 cells. This is a novel and unexpected finding since earlier studies suggest that KSHV reactivation would inhibit p53 function (Park, Seo et al. 2000, Gwack, Hwang et al. 2001, Nakamura, Li et al. 2001, Lee, Toth et al. 2009, Chudasama, Konrad et al. 2015). One explanation for our contradictory finding is that some of these referenced studies have been performed only with artificial overexpression methods (Gwack, Hwang et al. 2001, Nakamura, Li et al. 2001), whereas in our results we showed a p53 response in two independent lytic KSHV-infected cell lines (Fig 2E in III). On the other hand, it could be possible that p53 activity is carefully regulated during the lytic cycle so that in the early phase of reactivation, p53 is required for efficient replication, and in the later phase it needs to be inhibited by lytic proteins in order to prevent premature apoptosis. Indeed, there are indications that this is the case, since K8 was shown to interact with p53 in PEL cells 48 hours post TPA induction. Moreover, late lytic structural proteins ORF22, ORF25 and ORF64 were identified to inhibit p53-mediated transactivation of apoptotic genes (Park, Seo et al. 2000, Chudasama, Konrad et al. 2015). Therefore we wanted to further address the biological consequences of the p53 response at the early phase of lytic replication. 3.4 p53 is required at the early phase of the reactivation for efficient lytic replication To study the role of p53 in lytic replication we silenced p53 expression prior to the induction of reactivation. To this end, we first depleted p53 expression from iSLK.219 cells with lentivirusmediated shRNA for 48 hours and thereafter induced the cells for reactivation with TPA/Doxycycline (TPA/Dox) treatment. Reactivation was measured by RFP expression with automated fluorescence microscopy. This analysis showed that knockdown of p53 caused a remarkable decrease in the amount of cells undergoing lytic reactivation (Fig 3C-D in III). This was 58 RESULTS AND DISCUSSION further supported by a qPCR analysis for the expression of a set of lytic genes which showed impaired induction of lytic gene expression in p53-depleted BC-3 cells induced for reactivation by TPA or NaB treatment (Fig S4 C-D in III). To examine how stabilized p53 would influence the early steps of reactivation, we pre-treated the iSLK.219 cells with Nutlin-3 for 18 h, followed by TPA/Dox induction of reactivation for 24 h, and quantified RFP expression with automated fluorescent microscopy. Intriguingly, although Nutlin-3 treatment normally induces a strong p53 stabilization and G1 cell growth arrest in non-reactivated cells (Fig. S4 A-B and 6 C in III), under these conditions the cells did not arrest at G1, but rather progressed to G2 (Fig. 6 C in III). Importantly, we also demonstrated that Nutlin-3 synergizes with the TPA/Dox and Dox treatments and enhances the reactivation capacity of iSLK.219 cells (Fig 3A-B in III). Altogether, our studies revealed a distinct p53 response in lytic PEL cells, and strikingly, we found that efficient KSHV reactivation requires intact p53. Interestingly, human herpesviruses HSV-1, CMV, EBV and HIV-1 can utilize p53 to ensure efficient replication (Casavant, Luo et al. 2006, Pauls, Senserrich et al. 2006, Chang, Lo et al. 2008, Maruzuru, Fujii et al. 2013). However, the mechanisms used by these viruses appear different from KSHV as p53 has been reported to bind to viral lytic promoters. Therefore, it was somewhat surprising that we could not detect any p53 binding on the virus genome, a result which indicates that p53 does not participate directly in viral transcription (Fig. 2 B in III), and suggests that KSHV perhaps utilizes some of the p53 response genes to secure efficient replication. 3.5 p53 is required to induce p21Cip-1-mediated G2/M cell cycle arrest upon KSHV reactivation We next focused on one of the best-known p53 target genes, p21Cip-1, that we had already identified by ChIP-Seq and had found to be induced in the reactivated BC-3 and iSLK.219 cells (Fig. 2E in III). To address the requirement of p21Cip-1 in KSHV reactivation we silenced its expression by RNAi and showed that p21Cip-1 was required for efficient reactivation (Fig 3E-F in III). As p21Cip-1 is a Cdk inhibitor and can cause cell cycle arrest at G1 and G2 phases (el-Deiry, Tokino et al. 1993, Harper, Adami et al. 1993) we wanted to determine whether KSHV reactivation would lead to cell cycle arrest. For these studies we developed an automated, high-content image-based cell cycle analysis method (Fig. 5A and Fig. S6 in III). Cell cycle status of the cells in the lytic infection phase was investigated by indirect immunofluorescence (IF) using an antibody recognizing histone 3 phosphorylated on serine 10 (pH3 S10, Fig. 5A in III). Interestingly, this analysis revealed that the lytic iSLK.219 cells were arrested at the G2 phase of the cell cycle (Fig. 5 C-D in III). Depletion of p21Cip-1 from iSLK.219 cells prior to reactivation decreased the amount of cells in G2 arrest compared to si-Ctrl-transfected cells and increased the proportion of cells in M-phase (Fig 5 G in 59 RESULTS AND DISCUSSION III). As depletion of p21Cip-1 also significantly decreased viral reactivation (Fig 3E-F in III), these results indicate that KSHV requires p21 Cip-1 in order to arrest the reactivated cells at G2, which is necessary for efficient lytic replication. Induction of G2/M arrest has also been recognized with other herpesviruses. These studies show that a HHV-6B-encoded protein, DR6, induces a p53-independent G2 arrest and that an EBVencoded protein, LMP-1, induces a G2 arrest by upregulating 14-3-3 sigma and reprimo proteins which inactivate the Ckd1/CyclinB complex (Yeo, Mohidin et al. 2012, Schleimann, Hoberg et al. 2014). Thus our discovery shows for the first time that KSHV lytic infection also leads to the induction of G2/M arrest and that KSHV reactivation requires p53-dependent p21 Cip-1 activity to induce the G2 arrest, which probably functions to secure sufficient time to complete the viral lytic replication. This also explains why, unlike in many other human cancers, p53 is rarely mutated in the KSHV-associated malignancies (Katano, Sato et al. 2001). So what is the viral mechanism behind the reactivation induced, p21 Cip-1 mediated G2/M arrest? Interestingly v-cyclin can bypass a G1 arrest when complexed with CDK6 by phosphorylating p21Cip-1 and preventing its binding to CDK2 (Jarviluoma, Child et al. 2006). Since v-cyclin is not only expressed during the latent infection and its expression is actually increased upon lytic reactivation (Arias, Weisburd et al. 2014), it could be the mechanism behind the inactivation of the G1 checkpoint during lytic cycle. Importantly it has been shown that murine gammaherpesvirus 68 (γMHV-68) encoded v-cyclin is vital for lytic reactivation (van Dyk, Virgin et al. 2000). Therefore it remains to be seen if the KSHV v-cyclin also has a role in KSHV reactivation. Altogether results of this thesis project provide new insights as to how KSHV utilizes the p53 stress response in lytic reactivation. Importantly this finding could be one of the mechanisms how lytic KSHV infected cells develop drug resistance. Moreover, as previously shown in the case of Nutlin3, the restoration of p53 could not induce apoptosis or cell cycle arrest of in the lytic PEL cells (Sarek, Kurki et al. 2007, Sarek, Ma et al. 2013). Therefore, the results in this thesis that demonstrate the reactivation induced bypass of Nutlin-3 induced G1 arrest (Fig 6 C in III) provide a mechanism for the inability of Nutlin-3 to arrest or kill PEL cells. This is an important finding when considering the treatment modalities of KSHV induced cancers and could be one of the mechanisms behind drug resistance of the lytic KSHV infected cells. Another example on possible drug resistance of lytic KSHV infected cells is the study with c-MET inhibitor PF-2341066. Although c-MET inhibitor was shown to induce G2/M cell cycle arrest and DNA damage mediated apoptosis of PEL cells, this inhibitor was also inducing activation of p53 60 RESULTS AND DISCUSSION and minor lytic reactivation (Dai, Trillo-Tinoco et al. 2015). Therefore it could be possible that cMET inhibition is activating a p21Cip-1 mediated G2/M arrest which supports KSHV infected cells undergoing lytic replication and thus promotes cancer progression and could cause drug resistance during longer treatment periods. p21Cip-1 is pleiotropic protein as it is in the other hand inducing cell cycle arrest and apoptosis and thus preventing proliferation of cancer cells and acting as tumor suppressor, but in the other hand it can inhibit apoptosis by binding and inhibiting caspase-3, apoptosis signal-regulating kinase 1 (ASK1) and JNK and acting as oncogene (Asada, Yamada et al. 1999, Abbas and Dutta 2009, Cmielova and Rezacova 2011). P21Cip-1 is overexpressed in multiple cancers and is correlating with poor prognosis (Weiss 2003, Weiss, Borowsky et al. 2007, Abbas and Dutta 2009). Degree of p21Cip-1 function depends on the extent of the DNA damage, as in the case of low level DNA damage p21Cip-1 expression is increased inducing cell cycle arrest and inhibition of apoptosis and in the case of massive DNA damage p21Cip-1 expression is decreased causing induction of apoptosis (Cmielova and Rezacova 2011). Interestingly recent study showed that prolonged p21Cip-1 expression in p53-deficient cells caused replication stress and error-prone DNA repair could have prognostic and therapeutic implications for cancer (Galanos, Vougas et al. 2016). A subset of p53deficient cancer cells and tumours overexpressed nuclear p21Cip-1 and further studies with p53deficient cell line with inducible p21Cip-1 expression revealed that prolonged p21Cip-1 expression caused replication stress and error-prone DNA repair and resulted in development of chromosomal instability (Galanos, Vougas et al. 2016). P21Cip-1 inhibition could be explored as an anti-cancer approach and importantly studies with small molecule inhibitors of p21Cip-1 have been revealed their potential in treatment of chemo-resistant cancers (Park, Wang et al. 2008, Inoue, Hwang et al. 2011, Wettersten, Hee Hwang et al. 2013). It would be important to study the expression level of p21Cip-1 in KSHV associated cancers and the possibilities to inhibit the function of p21Cip-1 and target lytic KSHV infected cells and thus inhibit release of viral progeny. Treatment of PEL is challenging and patients often develop resistance against chemotherapy thus inhibition of p21Cip-1 could offer a therapeutic modality in treatment of PELp21Cip-1 61 RESULTS AND DISCUSSION 4. Characterization of the protein-protein interactome of v-cyclin and host cell proteins V-cyclin functions through interaction with cellular proteins like CDKs and most of the identified v-cyclin interactions are occurring via the CDK6 interaction. In the earlier studies v-cyclin interacting proteins have been identified either with transient, exogenous co-expression or immunoprecipitates from KSHV infected cell lines (Godden-Kent, Talbot et al. 1997, Li, Lee et al. 1997, Platt, Cannell et al. 2000, Chang and Li 2008). To reveal comprehensive interactome of vcyclin and cellular proteins a tetracycline inducible isogenic v-cyclin expressing human cell line was generated. 4.1 Generation of tetracycline inducible isogenic v-cyclin expressing human cell line To generate v-cyclin expressing cell line pcDNA5/FRT/TO/v-cyclin/SH/GW expression plasmids and pOG44 plasmid expressing Flippase (Flp) recombinase enzyme were co-transfected into HEK293 Flp-In cells (Invitrogen). Co-transfection induced Flp mediated homologous recombination between FRT sites of the cellular genome and expression plasmid (Figure 9A). Hygromycin resistance gene enables antibiotic selection of the isogenic v-cyclin cell clones. In these cells only one copy of v-cyclin gene is integrated into the host cell genome (Glatter, Wepf et al. 2009) and v-cyclin expression is repressed by tet-operator 2 (TetO2) that represses the promoter activity in the absence of tetracycline (Hillen and Berens 1994). This enables to adjust the v-cyclin expression to physiological expression levels and to avoid p53 induced growth arrest by continuous overexpression of v-cyclin. Cell lines generated in this study are named 293FlpIn-vcyc1 (vcyc1), 293FlpIn-vcyc2 (vcyc2) and 293FlpIn-Ctrl (Ctrl). Next optimal tetracycline concentration was titrated to obtain similar expression level of v-cyclin as in KSHV infected cells. Thus tetracycline was added 0, 8, 40, 200 and 1000 ng/ml to vcyc1 and vcyc2 cells and incubated 24 h prior the lysis. As a control whole cell lysates from non-tetracycline induced Ctrl cells, KSHV negative lymphoblastoid cell line (CZE) and KSHV positive PEL cell line (BC-3) were prepared. Lysate samples were Western Blotted using antibodies against v-cyclin, Hemagglutinin (HA) and tubulin. As illustrated in Figure 9B Ctrl and CZE cells did not express vcyclin or HA and as expected BC-3 cells expressed wild type v-cyclin, which, as expected, was migrating faster than the Strep- and HA-tagged v-cyclin of vcyc1 and vcyc2 cells. Linear increase in v-cyclin expression along with ascending concentration of tetracycline of the vcyc1 and vcyc2 cell lines was visible in both v-cyclin and HA blots (Figure 9B). Expression of v-cyclin was stronger in the vcyc1 cells than in the vcyc2 cells (Figure 9B v-cyclin and HA blots). Induction of the vcyc1 cells with 200 ng/ml of tetracycline resulted in similar v-cyclin expression level than in 62 RESULTS AND DISCUSSION BC-3 cells thus resembling the expression level in latently infected cells (Figure 9B). Expression of v-cyclin was also confirmed at mRNA level from the 200 ng/ml tetracycline induced vcyc1 and vcyc2 cell lines with the qPCR method (Figure 9C). V-cyclin expression was strongly induced with tetracycline and as seen at the protein level, v-cyclin expression was stronger in the vcyc1 cells than in the vcyc2 cells (Fiure 9C). Figure 9 Generation of the tetracycline inducible, isogenic v-cyclin expressing human cell line. Schematic illustration of the generation of the 293FlpIn-vcyc1 (vcyc1), 293FlpIn-vcyc2 (vcyc2) and 293FlpIn-Ctrl (Ctrl) cell lines (A). Western Blot analysis using antibodies to v-cyclin, HA and Tubulin of the 293FlpIn-Ctrl (Ctr), KSHV negative lymphoblastoid cell line (CZE) and KSHV positive PEL cell line (BC-3) and the tetracycline induced 293FlpIn-vcyc1 (vcyc1) and 293FlpIn-vcyc2 (vcyc2) cell lines. Specific tubulin band marked with asterisk (B). Quantified mRNA level of expressed ORF72 (v-cyclin) from 200 ng/ml tetracycline induced Ctrl, vcyc1 and vcyc2 cell lines with qPCR method (C). Although 200 ng/ml tetracycline induction led to expression of v-cyclin at similar levels as in the naturally infected cells, 1000 ng/ml concentration was decided to apply for the affinity purification to reach sufficient expression level to purify the bait protein efficiently. 4.2 Affinity purification of v-cyclin and its binding proteins To discover the v-cyclin protein-protein interaction network the vcyc1, vcyc2 and ctrl cells were induced 24h with 1000 ng/ml tetracycline and whole cell lysates were collected and subjected to 63 RESULTS AND DISCUSSION affinity purification (AP) utilizing the N-terminal strep-tag II peptide, which was immobilized to resin carrying streptavidin (Strep-Tactin) (Figure 10A). Samples were collected according to the AP procedure and resolved with SDS-PAGE and proteins were detected with silver staining to validate the quality of the purification procedure (Figure 10B.) Whole cell lysate sample (Lysate) showing the composition of the expressed proteins in the ctrl, vcyc1 and vcyc2 cells, flow through sample (Flowthrough) showing unbound proteins washed out from the Step-Tactin column and Strep-tag AP eluate sample (Eluate) showing purified proteins (Figure 10B). Silver gel shows that the bait protein was efficiently purified from the vcyc1 and vcyc2 lysates with Strep-tag AP (Eluate sample in Figure 10B, bait marked with asterisk). Abundance of the bait protein was higher in the vcyc1 eluate than in the vcyc2 eluate, which is in accordance with the result of v-cyclin and HA blotting in Figure 9B. Purification of the bait protein was confirmed also with Western Blotting of the lysates, flow through and the AP samples using an HA antibody (Figure 10C). The HA blot shows that ctrl lysate did not contain v-cyclin whereas from the vcyc1 and vcyc2 lysates v-cyclin was detected (Figure 10C, Lysate column). Importantly, from the flow through samples only a small amount of unbound bait protein was detected, indicating efficient binding of the bait protein to Strep-Tactin resin (Figure 10C, Flowthrough column). Eluate samples of vcyc1 and vcyc2 gave a strong HA signal, which was clearly stronger than in the lysate samples, indicating successful enrichment of the bait protein in the Strep-tag-AP (Figure 10C, Eluate column). Repeatedly, the vcyc1 eluate contained higher amount of v-cyclin bait protein than the vcyc2 eluate (Figure 10C, Eluate column). 64 RESULTS AND DISCUSSION Figure 10 Strep-tag affinity purification (AP) of the v-cyclin bait and the associating proteins. Schematic illustration of the strep-tag AP of the tetracycline induced Ctrl, vcyc1 and vcyc2 whole cell lysates (A). Validation of the AP process with silver gel. Whole cell lysates (Lysate), Flowthrough samples washed out from the Step-Tactin column (Flowthrough) and final eluates from the Strep-Tactin column of Ctrl, vcyc1 and vcyc2 were resolved with SDS-PAGE gel and proteins were detected by silver staining. Bait protein is marked with an asterisk. (B). Purification of the bait protein was confirmed also with Western Blotting (WB) of the lysates, Flowthrough and the AP eluate samples using an HA antibody (C). Double tag fusion protein generated in this study enables to utilize the tandem affinity purification (TAP) method. Although initially TAP was performed the bait recovery was not found sufficient (data not shown). TAP is more suitable for detection of stable protein-protein interactions (Chen and Gingras 2007) and as the v-cyclin mostly binds to its kinase partner(s), the other associating proteins are often only transiently bound, typically upon phosphorylation of a substrate. Therefore, the Strep-tag AP suited better for isolation of these protein complexes. Problem with the Strep-tag AP if compared to TAP is the higher probability of non-specific interactions. To identify high confidence protein interactome the Ctrl cell line and contaminant database compiled from 300 individual affinity purification pull down experiments were used to filter out non-specific interactions. 4.3 Identification of the v-cyclin interacting proteins by AP LC-MS/MS To pursue v-cyclin interacting proteins, purified protein complexes from the Strep-tag-AP of ctrl, vcyc1 and vcyc2 cell lines were analyzed with liquid chromatography mass spectrometry (LC65 RESULTS AND DISCUSSION MS/MS). Strep-tag-AP coupled to LC-MS/MS analysis was repeated twice. List of the v-cyclin interacting proteins is presented in table 4. Analysis of LC-MS/MS data revealed 14 v-cyclin interacting proteins of which six have been identified earlier (Table 4, known, marked with grey colour) and eight are previously unknown interactions (Table 4, novel, marked with yellow colour). Amongst the known interaction partners were three Cdks: Cdk2, Cdk5 and Cdk6 and three CDKIs: p16INK4A, p18INK4C and p27Kip1(Godden-Kent, Talbot et al. 1997, Li, Lee et al. 1997, Mann, Child et al. 1999, Jeffrey, Tong et al. 2000, Platt, Cannell et al. 2000, Kaldis, Ojala et al. 2001, Jarviluoma, Koopal et al. 2004). Cdk6 was detected with high peptide counts, which is in accordance with the current knowledge that v-cyclin primarily interacts with Cdk6 (Godden-Kent et. al 1997, Li et al. 1997, Meyerson & Harlow 1994, Ojala et al. 1999). Surprisingly, another well documented v-cyclin interaction partner Cdk4 was not amongst the peptides obtained from LC-MS/MS analysis, which could be due to a weak interaction of the v-cyclin with Cdk4 (Li, Lee et al. 1997). Additionally p16INK4A and p18INK4C have been reported to inhibit complex formation of v-cyclin and Cdk6 in HEK293 cells and in vitro (Jeffrey, Tong et al. 2000, Yoshioka, Noguchi et al. 2010). And as p16INK4A and p18INK4C were both pulled down with the v-cyclin bait it is possible that these CDKIs could inhibit the formation of v-cyclin/Cdk4 complex and thus this interaction was not detected from the LC-MS/MS analysis. Amongst the novel v-cyclin interacting proteins were two Cdks: Cdk1 and Cdk7, a deubiquitinating enzyme BRCA1 associated protein-1 (Bap1), a core chromatin histone H4, an anti-apoptotic paternally expressed gene 10 (Peg10), a nucleolar rRNA regulator and p53 activator/NF-κB repressor Myb-binding protein 1A (Mybbp1A), X-chromosome linked homologues of ubiquitin specific peptidase 9 protein (Usp9Y/X) and E3 ubiquitin ligase S-phase kinase associated protein 2 (Skp2) (Table 4). As Skp2 is targeting p27 Kip1 for ubiquitination and degradation (Nakayama, Nagahama et al. 2000), it is likely that it was pulled down together with the v-cyclin/Cdk6/p27 Kip1 complex and thus further validations of the Skp2 and v-cyclin interaction were not carried out in this study. However, validation of 6 other novel v-cyclin interacting partners was further pursued. 66 RESULTS AND DISCUSSION Table 4. List of v-cyclin interacting proteins determined by AP coupled LC-MS/MS analysis. Novel v-cyclin interacting proteins marked with yellow colour and known v-cyclin interacting proteins marked with grey colour. Protein Description Bap1 BRCA1 associated protein-1 Cdk1 Cyclin-dependent kinase 1 Cdk7 Cyclin-dependent kinase 7 H4 Histone H4 Mybbp1A Myb-binding protein 1A Peg10 Paternally expressed gene 10 Skp2 S-phase kinase-associated protein 2 Usp9Y/X Ubiquitin specific peptidase 9, Y and X-linked Cdk2 Cyclin-dependent kinase 2 Cdk5 Cyclin-dependent kinase 5 Cdk6 Cyclin-dependent kinase 6 INK4A p16 INK4C p18 Kip1 p27 Cyclin-dependent kinase inhibitor 2A Novelty Novel Known Cyclin-dependent kinase 4 inhibitor C Cyclin-dependent kinase inhibitor 1B 4.4 Validation of novel v-cyclin interacting proteins by Western Blot To validate the novel v-cyclin interacting proteins obtained from LC-MS/MS analysis lysates and the AP eluates of ctrl, vcyc1 and vcyc2 cell lines were Western Blotted with antibodies against endogenous Cdk1, Cdk7, Bap1, Histone H4, Mybbp1A, Usp9Y and Peg10. Endogenous protein levels of the analysed proteins were detected equally high from the ctrl, vcyc1 and vcyc2 cell lines, whereas Peg10 was detected at low levels and the vcyc1 and vcyc2 cells had higher expression levels than the ctrl cells (Figure 11, lysate samples). Interestingly Cdk1 was detected as a strong double band whereas Cdk7 displayed a strong and specific single band in the vcyc1 and vcyc2 APeluates (Figure 11, AP-Eluate samples). Bap1 was detected at low levels in the vcyc1 and vcyc2 AP-eluates. Histone H4, Mybbp1A, Usp9Y and Peg10 were not detected from the AP-eluate samples even after overnight exposure of the blot. Higher expression of the v-cyclin bait in the vcyc1 cells resulted in more efficient pull down of the validated interaction partners and importantly AP of the the ctrl did not pull down any of the validated proteins (Figure 11, Cdk1, Cdk7 and Bap1 blots of AP-Eluate samples). Thus interaction of v-cyclin and Cdk1, Cdk7 and Bap1 were confirmed by WB. 67 RESULTS AND DISCUSSION Figure 11 Western Blot validation of novel v-cyclin interacting proteins. To validate the novel v-cyclin interacting proteins obtained from LC-MS/MS analysis lysates and AP eluates of ctrl, vcyc1 and vcyc2 cell lines were Western Blotted with antibodies against endogenous Cdk1, Cdk7, Bap1, Histone H4, Mybbp1A, Usp9Y and Peg10. BAP1 is a tumour suppressor and a deubiquitinating enzyme, associated with processes such as DNA repair, cellular differentiation, cell cycle and proliferation (Wang, Papneja et al. 2016). BAP1 has been identified to deubiquitinate several proteins like the breast cancer type 1 susceptibility protein (BRCA1) and the host cell factor 1 (HCF-1) (Machida, Machida et al. 2009, Eletr and Wilkinson 2011, Carbone, Yang et al. 2013). As ubiquitination of the v-cyclin, which is not in complex with Cdk, has been shown to occur in HEK293 but not in BC-3 cells (Yoshioka, Noguchi et al. 2010), BAP1 and v-cyclin interaction can be connected to either deubiquitination of v-cyclin itself or its associating proteins. According to the LC-MS/MS and WB analysis of the AP samples Cdk1 was the most abundant protein amongst the novel v-cyclin interacting proteins. Moreover, the WB analysis indicated that v-cyclin was specifically pulling down two isoforms of the Cdk1 protein (Figure 11, Cdk1 blot of the vcyc1 and vcyc2 AP-eluates). Before mitosis Cdk1 is phosphorylated at Ser14 and Tyr15 68 RESULTS AND DISCUSSION residues by Wee1 and Myt1 kinases to inactivate the Cdk1/cyclinB1 complex and when the cell enters mitosis the Cdk1/cyclinB1 complex is de-phosphorylated by Cdc25 (Nigg 2001). Thus the lower band in the Cdk1 blot could represent a nonphosphorylated and active form of the Cdk1 protein. Interestingly v-cyclin/Cdk6 has been shown to phosphorylate Cdc25a and enhance its phosphatase activity (Mann, Child et al. 1999). Moreover, overexpression of v-cyclin dysregulates mitosis and induces centrosome amplification and multinucleation (Verschuren, Klefstrom et al. 2002, Koopal, Furuhjelm et al. 2007). Thus it is possible that the v-cyclin/Cdk6 is activating the Cdk1/cyclinB1 complex and in that way dysregulating the mitotic entry. Cdk7 forms the Cdk-activating kinase (CAK) complex with cyclin H and ménage a trois 1 (Mat1), which is required for the full kinase activity of CDK-cyclin complexes (Nigg 1996). Although the kinase activity of the v-cyclin/Cdk6 complex does not require the CAK complex, hyperphosphorylation of pRb and S-phase entry is fully accomplished only by the CAK phosphorylated v-cyclin/Cdk6 complex (Child and Mann 2001, Kaldis, Ojala et al. 2001). Additionally, the CAK complex is reported to protect the v-cyclin/Cdk6 complex from the p18 INK4C inhibition (Jeffrey, Tong et al. 2000). Thus the v-cyclin/Cdk7 interaction validated from the AP-LCMS/MS study could be due to the presence of the CAK-complex which supports the function of vcyclin/Cdk6 complex in promoting DNA replication. Another interesting finding has shown that prior to the Cdc25 catalysed activation, Cdk1 needs to undergo additional activating phosphorylation event by Cdk7 (Larochelle, Merrick et al. 2007). Thus in this case the v-cyclin Cdk7 interaction could have a role in the activation of Cdk1 and in the observed dysregulation of mitosis by v-cyclin. Although v-cyclin is reported to bind multiple Cdks it is reported, however, to form functional complexes only with Cdk2, Cdk6 and Cdk9 (Godden-Kent, Talbot et al. 1997, Mann, Child et al. 1999, Chang and Li 2008). This study identified the association of v-cyclin with two previously unknown Cdks: Cdk1 and Cdk7. Therefore in the light of these results it could be speculated that vcyclin might have a high binding affinity towards several Cdks and in that way acts like a Cdk recruiter that enables virus-induced steering of the cell cycle. 69 CONCLUSIONS AND FUTURE PROSPECTS CONCLUSIONS AND FUTURE PROSPECTS This study focused on how a cancer-associated herpesvirus manipulates the pro- and anti-apoptotic proteins of the host cell and utilizes the cell's stress response to secure efficient lytic replication. Furthermore this study discovered comprehensive protein interactome of v-cyclin and cellular proteins. Research in this thesis identified that virally encoded miRNAs inhibit apoptosis by silencing the expression of an effector caspase 3 in the latent KSHV-infected cells. In this study we were able to discover a new cellular target of KSHV-encoded miRNAs and to uncover a novel anti-apoptotic mechanism of this tumor virus. In the future it would be interesting to determine whether TinyLNA oligos inhibiting miR-K12-1, K12-3 and K12-4-3p miRNAs sensitize KSHV-associated tumors to etoposide or doxorubicin-induced apoptosis in a mouse model, which could provide new leads to treat KS and PEL. During the KSHV latency phase, p53 is kept under control by LANA and MDM2 to allow cell proliferation and inhibit apoptosis. Results in this thesis provide new insights as to how KSHV can cope with and utilize the p53 stress response during lytic reactivation. Accordingly, these results demonstrate that at the early steps of the reactivation cascade, p53 is required to induce p21 Cip-1mediated G2/M cell cycle arrest, probably to ensure adequate time for sufficient completion of the lytic replication cycle. It remains to be seen whether cellular stresses such as hypoxia and oxidative stress can activate the p53 response and aid the virus in efficiently completing the lytic cycle. However, the p53 response can also lead to apoptosis during the lytic cycle and thus prevent release of the viral progeny from the stressed cell. Nevertheless, premature apoptosis can be prevented by anti-apoptotic viral and cellular proteins inhibiting the pro-apoptotic downstream targets of p53. Importantly, our studies suggest that the survival of KSHV-infected cells is dependent on the cellular anti-apopotic Mcl-1 protein. Furthermore, this thesis identified a potent anti-viral drug to specifically target the lytic KSHV-infected cells. This study discovered that a pan Bcl-2 inhibitor, Obatoclax, targeting both Mcl-1 and other Bcl-2 proteins, induces cell death in lytic phase KSHVinfected cells. Hence, Obatoclax could be a promising new drug candidate to treat KSHVassociated cancers. To study potential anti-viral effect of Obatoclax further it would be important to address if Obatoclax can inhibit the release of viral progeny and test its potential anti-tumor effect in PEL xenograft models. With help of the isogenic and inducible v-cyclin expressing human cell line this study was able to identify 8 novel v-cyclin interaction partners from which Cdk1, Cdk7 and Bap1 were successfully 70 CONCLUSIONS AND FUTURE PROSPECTS validated. Although the AP-LC-MS/MS method gives an easy and reliable snapshot of proteinprotein complex networks, it cannot distinguish direct interactions from the detailed biochemical features of the protein complexes. To further identify the composition of the v-cyclin interacting protein complexes, additional biochemical approaches such as size-exclusion chromatography, immunoprecipitation or fluorescence resonance energy transfer (FRET) should be applied. Also functional experiments to identify the biological role of these novel interactions for the virus-host interaction and life cycles of KSHV and in the context of the other viral proteins would be interesting to pursue. 71 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS This study was performed at the Institute of Biotechnology and at the Faculty of Medicine Research Program Unit, Biomedicum Helsinki. I wish to thank Helsinki Biomedical Graduate Program for their financial support and interesting courses and especially student council activity. This research work has also been supported by grants from the Foundation on Viral Diseases, K.Albin Johanssons Foundation, Instrumentarium Research Foundation and Research Foundation of the University of Helsinki. I would like to thank my supervisors Professor Päivi Ojala and Dr. Giuseppe Balistreri for their scientific guidance and for the opportunity to perform interesting research and to develop into a critical scientist. I thank my thesis committee members Professor Olli Jänne and Docent Carina Holmber-Still for their comments and suggestion during this research. I wish to express my gratitude to my thesis’ pre-examiners Docent Sisko Tauriainen and Professor Jukka Westermarck for their valuable comments. I greatly acknowledge Ryan Murray for rapid and professional assistance with linguistic revision. I am grateful for my excellent collaborators: Dr. Sébastien Pfeffer and Dr. Guillaume Suffert for sharing the data and samples for miRNA project; Dr. Denis Kainov, Dr. Laura Kakkola and Dr. Oxana Denisova for the collaboration in ABT-263 and Obatoclax project; Professor Olli Kallioniemi and Dr. Juha Rantala for co-operation in CSMA siRNA sceen; Docent Markku Varjosalo for LC-MS/MS analysis; Professor Jussi Taipale and M.Sc. Mikko Turunen for performing ChIP-seq analysis, Professor Sampsa Hautaniemi and BSc. Lauri Lyly for analysis of ChiP-seq data, Professor Marikki Laiho and Dr. Karita Peltonen for providing reagents and technical help in MDM2 project, Professor Ilkka Julkunen for providing facilities for baculovirus production for siRNA screen project. I have been very fortunate to work with Ojalalab group members. I am so grateful that you have created such a nice, supporting and encouraging atmosphere in the lab and I have shared countless fun moments with you guys. I would like to acknowledge also the lab managers Jenny Bärlund, Anne Aarnio and Sari Tynkkynen for performing excellent technical support for the lab. Especially I would like to thank my “gangsta girls” Jenni Vaaksio, Elisa Kaivanto and Monica Bhaskar for sharing all the ups and downs of the research work and life in general with me, you made my path so much easier and I am so grateful of your friendship. 72 ACKNOWLEDGEMENTS I am deeply grateful for my dear family. You have provided me with endless support and encouragement to carry on with this road. My unending gratitude goes to my beloved mother Anja and my sister and best friend Elisa for their limitless love and support. I could not have done without you. I am grateful for all of my friends you have been helping me to relax and enjoy my life. Johanna and Miia we have shared so many precious moments together since the freshmen year at the University of Kuopio, you are so dear to me. And thank you Johanna for all those therapeutic discussions of PhD and beyond that. I would like to thank Mikko for your friendship and support along the years; you are like brother to me. I would like to thank also the members of “Aitoa Menoa” team for those exciting and fun bar quizzes. Thank you Monica for those fun nights at Teerenpeli and memorable trips to Venice and Bern, with you I have always such a great time. Thank you Elisa for your support and great discussions about future and life in general, it has been really therapeutic for me. And especially I would like to acknowledge Jenni for helping me with the final formatting of the thesis manuscript and supporting me during these last weeks. I have shared so many good memories with you and I am really grateful for those moments, you are a true friend. I would like to express my deepest gratitude to my love and best friend Markus. Thank you for your strong support and gentle coaching during my writing process. I am so grateful for your encouraging words at the times when I didn’t believe in myself. I cannot thank you enough that you have stood by me all this time and I am really happy to have you in my life. Johanna Viiliäinen Helsinki, September 2016 73 BIBLIOGRAPHY BIBLIOGRAPHY Abbas, T. and A. Dutta (2009). "p21 in cancer: intricate networks and multiple activities." Nat Rev Cancer 9(6): 400414. Akula, S. M., P. P. Naranatt, N. S. 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