Oncolytic H‐1 Parvovirus Ns1 Protein Identifying and Characterizing New Transcriptional and Post‐translational Regulatory Elements

.................................................................................................................................................... 7 PROLOGUE ONCE UPON A TIME (IMMEMORIAL).............................................................................................. ONCE UPON A TIME (IMMEMORIAL) .................................................................................................................. 8 INTRODUCTION BOOK I. THE PARVOVIRIDAE STORY ........................................................................................ BOOK I. THE PARVOVIRIDAE STORY .................................................................................................................... 9 Part 1. Family portrait of a killer .................................................................................................................... 9 Chapter 1. The family ................................................................................................................................................. 10 Chapter 2. The subfamilies ........................................................................................................................................ 11 Chapter 3. The genera ............................................................................................................................................... 12 Chapter 4. The species ............................................................................................................................................... 13 Part 2. Anatomy of the killer ........................................................................................................................ 14 Chapter 1. Organization and structure of H‐1PV genome ......................................................................................... 15 Paragraph 1. General features .............................................................................................................................. 15 Paragraph 2. The right‐hand end hairpin .............................................................................................................. 16 Paragraph 3. The left‐hand end hairpin ................................................................................................................ 16 Description of the hairpin ................................................................................................................................ 16 Importance of the left‐end hairpin’s asymmetry ............................................................................................. 17 Additional functions of the left‐end hairpin ..................................................................................................... 18 Chapter 2. The viral particle ....................................................................................................................................... 18 Chapter 3. Associated diseases .................................................................................................................................. 19 BOOK II. THE H‐1 PARVOVIRUS STORY .............................................................................................................. 20 Part 1. Typical day of the killer ..................................................................................................................... 20 Chapter 1. The virus enters the cell... ........................................................................................................................ 20 Chapter 2. ...then heads the nucleus through the endosomal pathway... ................................................................. 21 Chapter 3. ...before uncoating, which makes the viral DNA available for... ............................................................... 23 Chapter 4. ...rolling‐hairpin replication... ................................................................................................................... 24 te l-0 08 26 93 6, v er si on 1 28 M ay 2 01 3 Chapter 5. ... as well as transcription... ...................................................................................................................... 25 Chapter 6. ...in order to create new virions ............................................................................................................... 26 Chapter 7. ...that are transported and release back to the extracellular matrix. ....................................................... 27 Part 2. Modus operandi of the killer ............................................................................................................. 28 Chapter 1. Oncosuppression ...................................................................................................................................... 29 Chapter 2. Oncotropism ............................................................................................................................................ 31 Paragraph 1. The P4 connection ........................................................................................................................... 31 Paragraph 2. Beyond transcription ....................................................................................................................... 33 Paragraph 3. New findings .................................................................................................................................... 33 Paragraph 4. The unreachable definition of oncotropism .................................................................................... 35 Chapter 3. Oncolysis .................................................................................................................................................. 36 Part 3. Redemption of the killer .................................................................................................................... 37 Chapter 1. Oncolytic viruses as clinical anticancer agents. ........................................................................................ 37 Chapter 2. H‐1PV as an anticancer therapy: interactions with the immune system and clinical developments. ...... 39 Paragraph 1. Antiviral immune responses ............................................................................................................ 40 Paragraph 2. Antitumor vaccination and H‐1PV adjuvant effect .......................................................................... 40 Paragraph 3. Direct and indirect interactions with the immune system .............................................................. 41 Paragraph 4. Immunomodulation by engineered infectious H‐1PV. .................................................................... 42 Paragraph 5. Clinical developments. ..................................................................................................................... 42 BOOK III. THE NS PROTEIN STORY...................................................................................................................... 45 Part 1. NS2, the shy arm of the killer. ........................................................................................................... 45 Part 2. NS1, the versatile arm of the killer. ................................................................................................... 46 Chapter 1. NS1 involvement throughout H‐1PV life cycle. ........................................................................................ 47 Paragraph 1. Involvement in viral DNA amplification ........................................................................................... 47 Paragraph 2. Involvement in viral and cellular transcription ................................................................................ 48 Paragraph 3. Involvement in viral cytotoxicity ...................................................................................................... 49 Necrosis ............................................................................................................................................................ 49 Cytoskeleton‐related cell death ....................................................................................................................... 50 Apoptosis ......................................................................................................................................................... 51 Cell cycle arrest ................................................................................................................................................ 52 Chapter 2. Different levels of NS1 regulation. ........................................................................................................... 52 Paragraph 1. Posttranslational level of NS1 regulation ......................................................................................... 53 Paragraph 2. Spatial level of NS1 regulation ......................................................................................................... 54 Paragraph 3. Temporal level of NS1 regulation .................................................................................................... 54 BOOK IV. THE NARROW ESCAPE STORY ............................................................................................................ 55 Part I. Apoptosis, the first molecular barrier raised to eradicate viruses. .................................................... 55 Part 2. Apoptosis, the first molecular barrier viruses have learnt to handle. ............................................... 56 Review. Caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis. ......... 57 AIMS OF THE WORK ........................................................................................................................................ SHOOTING AN ARROW INTO THE AIR AND, WHERE IT LANDS, PAINTING A TARGET ........................................ 58 te l-0 08 26 93 6, v er si on 1 28 M ay 2 01 3 RESULTS & DISCUSSION ................................................................................................................................... Article I. Different involvement in the viral life cycle of the Y‐boxes within H‐1 parvovirus P4 promoter. ... 59 Discussion about Article I. ............................................................................................................................ 68 About results related to standard P4 promoter analysis by transactivation assays .................................................. 68 About results related to the study of H‐1PV molecular clones carrying modified Y‐boxes. ...................................... 69 Article II. Caspase cleavage of H‐1 parvovirus NS1 protein in non transformed cells generates fragments with dominant negative functions. ............................................................................................................... 71 Discussion about Article II. ........................................................................................................................... 92 Induction of apoptosis in H‐1PV‐infected non transformed cells: the result of an immune antiviral response ? ..... 92 EPILOGUE ........................................................................................................................................................ AND THEY KILLED HAPPILY EVER AFTER ............................................................................................................ 95 REFERENCES ................................................................................................................................................ 97 ANNEXES ................................................................................................................................................... 111 te l-0 08 26 93 6, v er si on 1 28 M ay 2 01 3