Characterization of molecular targets for differential regulation of the type I and III interferon induction and signalling pathways by rotavirus NSP1 Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Gennaro Iaconis September 2018 Table of contents TABLE OF CONTENTS ............................................................................................................................... 1 LIST OF FIGURES ...................................................................................................................................... 6 LIST OF TABLES ........................................................................................................................................ 8 DECLARATION ....................................................................................................................................... 10 ABSTRACT ............................................................................................................................................. 11 1 INTRODUCTION ........................................................................................................................... 12 1.1 Rotavirus ................................................................................................................. 12 1.1.1 Historical Background ......................................................................................... 12 1.1.2 Classification ....................................................................................................... 13 1.1.2.1 Reoviridae ...................................................................................................... 13 1.1.2.1.1 Rotavirus ................................................................................................ 14 1.1.3 Epidemiology ...................................................................................................... 16 1.1.4 Pathogenesis and pathology ............................................................................... 18 1.1.5 Rotavirus vaccine ................................................................................................ 19 1.1.6 Virion Structure ................................................................................................... 20 1.1.7 Genome organization ......................................................................................... 24 1.1.8 Gene-protein coding assignments ...................................................................... 25 1.1.9 Rotavirus proteins ............................................................................................... 26 1.1.9.1 Rotavirus structural proteins (VPs) ................................................................ 26 1.1.9.1.1 VP1 (core) ............................................................................................... 26 1.1.9.1.2 VP2 (core) ............................................................................................... 26 1.1.9.1.3 VP3 (core) ............................................................................................... 27 1.1.9.1.4 VP4 (outer capsid protein) ..................................................................... 27 1.1.9.1.5 VP6 (inner capsid protein) ..................................................................... 28 1.1.9.1.6 VP7 (outer capsid protein) ..................................................................... 28 1.1.9.2 Rotavirus non-structural proteins (NSPs) ...................................................... 29 1.1.9.2.1 NSP1 ....................................................................................................... 29 1.1.9.2.2 NSP2 ....................................................................................................... 31 1.1.9.2.3 NSP3 ....................................................................................................... 33 1.1.9.2.4 NSP4 ....................................................................................................... 33 1.1.9.2.5 NSP5 ....................................................................................................... 35 1.1.9.2.6 NSP6 ....................................................................................................... 35 1.1.10 Rotavirus replication cycle .................................................................................. 37 1.1.10.1 Virus attachment ......................................................................................... 39 1.1.10.2 Penetration .................................................................................................. 40 1.1.10.3 Uncoating ..................................................................................................... 41 1 1.1.10.4 Transcription ................................................................................................ 41 1.1.10.5 Translation ................................................................................................... 42 1.1.10.6 Viroplasms formation and genome replication ........................................... 43 1.1.10.7 Virion assembly, maturation and release .................................................... 44 1.2 Interferon ................................................................................................................ 47 1.2.1 Toll-like receptors ............................................................................................... 52 1.2.2 RIG-I receptors .................................................................................................... 56 1.2.2.1 RIG-I ............................................................................................................... 57 1.2.2.2 MDA5 ............................................................................................................. 57 1.2.2.3 LGP2 ............................................................................................................... 57 1.2.3 MAVS .................................................................................................................. 58 1.2.4 Regulation of the IFN: promoters and induction ................................................ 59 1.2.5 Structure of the promoter of Type I IFN ............................................................. 59 1.2.6 Structure of the promoter of Type III IFN ........................................................... 59 1.2.7 JAK / STAT pathway ............................................................................................ 64 1.3 Modulation of the host innate immunity by rotavirus NSP1 .................................... 65 1.3.1 IRFs ...................................................................................................................... 66 1.3.2 RIG-I, MDA5 and MAVS ....................................................................................... 67 1.3.3 NF-κB ................................................................................................................... 67 1.3.4 STAT1 and STAT2 ................................................................................................ 68 1.4 Aims ........................................................................................................................ 70 2 MATERIAL AND METHODS ........................................................................................................... 72 2.1 Cell Culture .............................................................................................................. 72 2.1.1 Cell lines .............................................................................................................. 72 2.1.2 Maintenance of cells in tissue culture ................................................................ 73 2.1.3 Maintenance of stable cells in tissue culture...................................................... 73 2.1.4 Transfection ........................................................................................................ 73 2.2 Viruses .................................................................................................................... 74 2.2.1 Sendai virus ......................................................................................................... 74 2.2.2 Classical Swine fever Virus .................................................................................. 74 2.2.3 CSFV infection of PK-15 cell lines ........................................................................ 74 2.2.4 Modified Vaccinia Ankara –T7 polymerase (MVA-T7) ........................................ 74 2.3 Cloning and plasmid preparation ............................................................................ 75 2 2.3.1 Polymerase chain reaction (PCR) ........................................................................ 75 2.3.2 Agarose gel electrophoresis ................................................................................ 76 2.3.3 Gel purification of DNA ....................................................................................... 76 2.3.4 Restriction enzyme digestion of DNA ................................................................. 76 2.3.5 Dephosphorylation of vector DNA ...................................................................... 77 2.3.6 DNA ligation ........................................................................................................ 77 2.3.7 Transformation ..................................................................................................