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Investigating the Role of Host TTR-Rbps During SFV4 and MHV-68 Infection

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Investigating the Role of Host TTR-Rbps During SFV4 and MHV-68 Infection Investigating the role of host TTR-RBPs during SFV4 and MHV-68 infection Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Jamie Casswell October 2019 Contents Figure Contents Page……………………………………………………………………………………7 Table Contents Page…………………………………………………………………………………….9 Acknowledgements……………………………………………………………………………………10 Abbreviations…………………………………………………………………………………………….11 Abstract……………………………………………………………………………………………………..17 1. Chapter 1 Introduction……………………………………………………………………………19 1.1 DNA and RNA viruses ............................................................................. 20 1.2 Taxonomy of eukaryotic viruses ............................................................. 21 1.3 Arboviruses ............................................................................................ 22 1.4 Togaviridae ............................................................................................ 22 1.4.1 Alphaviruses ............................................................................................................................. 23 1.4.1.1 Semliki Forest Virus ........................................................................................................... 25 1.4.1.2 Alphavirus virion structure and structural proteins ......................................................... 26 1.4.1.3 Alphavirus non-structural proteins ................................................................................... 29 1.4.1.4 Alphavirus genome organisation ...................................................................................... 31 1.4.1.5 Cis-acting RNA elements in Alphavirus RNA ..................................................................... 32 1.4.1.6 Alphavirus replication in mammalian cells ....................................................................... 34 1.5 Herpesviridae ......................................................................................... 39 1.5.1 Alphaherpesvirinae and Betaherpesvirinae ............................................................................. 39 1.5.2 Gammaherpesvirinae ............................................................................................................... 39 1.5.2.1 MHV-68 ............................................................................................................................. 40 1.5.2.3 Genome and virion structure of gammaherpesviruses .................................................... 40 1.5.2.4 Replication of gammaherpesviruses ................................................................................. 41 2 1.6 Mammalian mRNA ................................................................................. 42 1.6.1 Mammalian mRNA maturation ................................................................................................ 42 1.6.2 mRNA export in mammalian cells ............................................................................................ 43 1.6.3 mRNA decay ............................................................................................................................. 44 1.6.4 Turnover and Translation RNA-binding proteins (TTR-RBPs) .................................................. 45 1.6.4.1 HuR .................................................................................................................................... 45 1.6.4.2 PABPC ................................................................................................................................ 46 1.7 Virus inhibition and manipulation of RNA decay pathways .................... 47 1.7.1 Host shut-off ............................................................................................................................ 47 1.7.1.1 muSOX............................................................................................................................... 48 1.7.2 Virus utilisation of host TTR-RBPs ............................................................................................ 48 1.7.2.1 HuR in alphavirus infection ............................................................................................... 49 1.7.2.2 PABPC in viral infection ..................................................................................................... 50 1.8 General Aims .......................................................................................... 50 2.1 Cell culture ............................................................................................. 52 2.1.1 Mammalian cell culture maintenance ..................................................................................... 52 Baby hamster kidney fibroblasts (BHK-21 cell line) ...................................................................... 52 2.1.2 Subculture of mammalian cells................................................................................................ 53 2.1.3 Cryopreservation and resuscitation of cell stocks ................................................................... 53 2.2 Generation of recombinant SFV4 infectious complementary DNA plasmids ....................................................................................................... 54 2.3. Generation of virus stocks ..................................................................... 56 2.3.1. Generation of rSFV4 stocks ..................................................................................................... 58 2.3.1.1. In vitro transcription of SFV4(3F)-ZsGreen ...................................................................... 58 2.3.1.2 Transfection of rSFV4 encoding nucleic acid for virus propagation ................................. 58 2.3.1.3. Purification of rSFV4 viruses ............................................................................................ 59 2.3.2 Murine gammaherpesvirus-68 stocks ..................................................................................... 59 3 2.4 General Virus Techniques ....................................................................... 60 2.4.1 Infections ................................................................................................................................. 60 2.4.2 Virus titration by Plaque Assay ................................................................................................ 61 2.5 General bacterial techniques .................................................................. 62 2.5.1 Bacterial Culture ...................................................................................................................... 62 2.5.2 Preparation and use of glycerol stocks .................................................................................... 62 2.5.3 Transformation of competent E.coli ........................................................................................ 62 2.5.4 Plasmid DNA extraction from bacterial cultures ..................................................................... 63 2.6 General nucleic acid and molecular techniques ...................................... 65 2.6.1 Extraction of RNA from mammalian cells ................................................................................ 65 2.6.2 Nuclear and Cytoplasmic Fractionation followed by RNA extraction ...................................... 65 2.6.3 DNase Treatment of RNA samples ........................................................................................... 66 2.6.4 RNA clean-up of DNase Treated RNA samples ........................................................................ 66 2.6.5 Reverse Transcription of RNA samples .................................................................................... 66 2.6.6 Nucleic acid quantification ...................................................................................................... 67 2.6.7 Restriction Digest and FastAP treatment ................................................................................ 67 2.6.8 DNA Ligations ........................................................................................................................... 67 2.6.9 Gel Electrophoresis .................................................................................................................. 68 2.6.10 Extraction of DNA from gels .................................................................................................. 68 2.6.11 Blunt-end TOPO cloning......................................................................................................... 68 2.6.12 Standard PCR ......................................................................................................................... 69 2.6.13 Colony PCR ............................................................................................................................. 70 2.6.14 Sequencing ............................................................................................................................. 70 2.7 Genetic reversion analysis of viral mutants ............................................ 70 2.7.1 Genetic reversion analysis of rSFV4-URE/CSE viruses ............................................................. 70 2.7.2 Analysis of genetic reversion of MHV-68 ORF37stop virus ..................................................... 71 2.8 Immunostaining and Confocal Microscopy ............................................. 71 4 2.9 In vitro characterisation of rSFV4 URE/CSE mutants ............................... 74 2.9.1
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