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Antiviral Potential Coupled to Genome Stability: the Multifaceted Roles of DDX3X Protein

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Antiviral Potential Coupled to Genome Stability: the Multifaceted Roles of DDX3X Protein Dipartimento di Biologia e Biotecnologie “L. Spallanzani” Consiglio Nazionale delle Ricerche, Istituto di Genetica Molecolare Luigi Luca Cavalli-Sforza Antiviral potential coupled to genome stability: the multifaceted roles of DDX3X protein Valentina Riva Dottorato di Ricerca in Genetica, Biologia Molecolare e Cellulare Ciclo XXXII – A.A. 2016-2019 Dipartimento di Biologia e Biotecnologie “L. Spallanzani” Consiglio Nazionale delle Ricerche, Istituto di Genetica Molecolare Luigi Luca Cavalli-Sforza Antiviral potential coupled to genome stability: the multifaceted roles of DDX3X protein Valentina Riva Supervised by Prof. Giovanni Maga Dottorato di Ricerca in Genetica, Biologia Molecolare e Cellulare Ciclo XXXII – A.A. 2016-2019 Abstract The human RNA helicase DDX3X is a real multifaceted enzyme. Like all the other DEAD-box proteins of the same family, DDX3X participates into different steps of RNA metabolism. Moreover, DDX3X is one of the actors of cell cycle regulation, innate immunity and apoptosis processes. Our group started to look at DDX3X as an interesting protein since it has primary roles in viral infections and tumor development too. In the context of viral infections, DDX3X possesses dual roles: it acts as an antiviral or proviral factor regulating viral replication at different levels (regulation of genome duplication and/or gene expression and host innate immunity activation). From these observations, it came the idea to use DDX3X as a possible therapeutic target to inhibit a function essential for the viral replication, but dispensable for the human cell. In collaboration with the University of Siena (Prof. Maurizio Botta), we developed some inhibitor molecules able to recognize two different DDX3X pockets: the helicase binding pocket and the unique motif of DDX3X. Both two compounds families showed selectivity and no toxicity in cells; even more interesting, our molecules showed considerable broad-spectrum antiviral effects being able to suppress both the replication of WNV and DENV-2 viruses in infected cells. The development of DDX3X-specific inhibitor molecules in the context of different viral infections is just one of the two projects that I followed during my PhD internship. In parallel, I shifted my attention on the characterization of this enzyme in the context of DNA damage response. Published data have already implicated DDX3X in the resolution of RNA/DNA hybrids and RNA secondary structures, as well as in the degradation of RNAs. Moreover, unpublished data from our lab demonstrated also a possible exoribonuclease activity of DDX3X in addition to the already well-known helicase/ATPase activities. Trying to envision a physiological relevance of this activity, we focused on the possible situations in which RNA/DNA hybrids could be present into the cell. Ribonucleotides (rNMP) are the most abundant type of DNA damage and all the cells have specific enzymes able to remove them avoiding deleterious consequences. This removal activity is essential and it is primarily performed by the ribonucleotide excision repair (RER) pathway. RNaseH2 is believed to be the only enzyme able to incise single rNMPs within a DNA strand activating an error-free RER pathway. Surprisingly, our data demonstrated that DDX3X is able to promote RER initiation, thereby suggesting a possible new role of this enzyme also in genome stability. 3 Acknowledgements I want to express my sincere gratitude to my supervisor Giovanni Maga for the opportunity to be part of his laboratory and to follow what for me is” the most beautiful project of the world”. Nothing would have been possible without my first lab-teacher Anna, that time has transformed into a friend. My acknowledgements to Doctor Silvio Spadari as well, always optimistic and able to lighten even the heaviest days. Two special thanks to other two professors not really part of my laboratory, Martina and Simone: without your advices I wouldn’t arrive at the end of this manuscript. During years a lot of people were part of the big “Lab 500”: thanks to everyone, for every single teaching and every single laugh. A very special thanks to all my students: DDX3 family is something that it will remain into my heart. Thanks to all my friends, for supporting me in all difficult moments and for being at my side now and over the years. Thanks to my family without which I wouldn’t be what I am. Thanks to my “Quail”: anyone could understand better than you the complicated world of Biology. Thank to be my complicit, I hope you’ll continue to be it for long long time. 4 Abbreviations AGS Aicardi-Goutier syndrome BAdV-3 Bovine Adenovirus 3 BVDV Bovine Viral Diarrhea virus CK1ɛ Casein kinase 1 epsilon CRM1 Chromosomal Maintenance 1 or Exportin 1 DSBs Double Strand Breaks DBY DEAD-box Y RNA helicase 3 DDX1 DEAD-box helicase 1 DDX3X DEAD-box helicase 3 X-linked DDX3Y DEAD-box helicase 3 Y-linked DENV Dengue virus Dicer Endoribonuclease Dicer Dox Doxycycline ds Double strand Dvl1 Segment polarity protein dishevelled homolog DVL- 1 Dvl2 Segment polarity protein dishevelled homolog DVL- 2 eIF3 Eukaryotic translation initiation factor 3 ENKTL-NT Extranodal NK/T cell lymphoma, nasal type or NKTCL Exo1 Exoribonuclease 1 Fen1 Flap Endonuclease 1 HBV Hepatitis B Virus HCMV Human cytomegalovirus HCV Hepatitis C Virus HIF-1α Hypoxia-inducible factor 1-alpha HIV-1 Human Immunodeficiency virus type 1 HPV Human papilloma virus HSV-1 Herpes simplex virus type 1 IAV Human Influenza A virus IC50 Half maximal inhibitory concentration IFN Interferon IKKɛ Inhibitor of nuclear factor kappa-B kinase subunit epsilon 5 IRF3 Interferon regulatory factor 3 JEV Japanese encephalitis virus JUNV Junin virus K7 Protein K7 KRAS GTPase KRas LASV Lassa virus LCMV Lymphocytic choriomeningitis virus MDM2 E3 ubiquitin-protein ligase Mdm2 MITF Microphthalmia-associated transcription factor MNV Murine Norovirus MOI Multiplicity Of Infection NES Nuclear export signal NF-kB Nuclear factor kappa-light-chain-enhancer of activated B cells NLS Nuclear localization signal NP Nucleoprotein Npro Genome polyprotein NS1 Non-structural protein 1 NS3 Non-structural protein 3 NS5 Non-structural protein 5 NSC cells Non-silenced cells nsP3 Non-structural polyprotein 3 nt nucleotide NZ51 Ring-expanded nucleoside analogue OSCC Oral squamous cell cancer PB1-F2 Influenza A proapoptotic protein PCNA Proliferating Cell Nuclear Antigen PFU Plaque Forming Unit PLK1 Serine/threonine-protein kinase PLK1 Pols Polymerases RER Ribonucleotide Excision Repair RIG-I RIG-I-like receptor 1; Probable ATP-dependent RNA helicase DDX58 RK33 diimidazo[4,5-d:40,50-f]-[1,3]diazepine RNaseH1/2 Ribonuclease H 1/2 RNaseR Ribonuclease R rNMPs Ribonucleotides triphosphates RS Arginine-Serine dipeptide SF1-6 Superfamilies 1-6 SGs Stress granules 6 ShDDX3 DDX3X KD cells cells Srs2 ATP-dependent DNA helicase SRS2 ss Single strand Tat Transactivating regulatory protein TBK1 Serine/threonine-protein kinase TBK1 TLC Thin-Layer Chromatography TNM Tumor Node and Metastasis Top1 Topoisomerase 1 VACV Vaccinia virus VEEV Venezuelan Equine Encephalitis virus WNV West Nile virus 7 Contents Contents Abstract .......................................................................................................... 3 Acknowledgements ........................................................................................ 4 Abbreviations ................................................................................................. 5 Contents ......................................................................................................... 8 1. Introduction ............................................................................................. 10 2. Review of the literature ........................................................................... 11 2.1 Human DDX3 genes ........................................................................... 11 2.2 DDX3X protein .................................................................................. 11 2.3 Cellular localization of DDX3X in normal and tumor cells .............. 20 2.4 DDX3X and cell proliferation regulation .......................................... 21 2.5 DDX3X in innate immunity and stress response ................................ 22 2.6 DDX3X and cancer ............................................................................ 23 2.7 DDX3X as an anticancer drug target ................................................ 28 2.8 Genomic stability: DDX3X in the response of ribonucleotide incorporation ........................................................................................... 30 2.9 DDX3X and viral infections ............................................................... 34 2.10 DDX3X as a target for antiviral therapies ....................................... 42 3. Aims of the research ................................................................................ 45 4. Materials and methods ............................................................................ 47 4.1 DDX3X AS A PRIME CELLULAR TARGET TO FIGHT OLD AND EMERGING VIRUSES ............................................................................. 47 4.1.1 Chemistry .................................................................................... 47 4.1.2 Proteins production and purification ........................................... 47 4.1.3 Helicase assay based on Fluorescence Resonance Energy Transfer (FRET) ................................................................................... 48 4.1.4 ATPase assay .............................................................................
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