Stokes, Samuel (2018) Determining the role of Aedes aegypti host SUMOylation in suppressing arbovirus replication. PhD thesis. https://theses.gla.ac.uk/39054/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] S Stokes, 2018 1 Determining the role of Aedes aegypti host SUMOylation in suppressing arbovirus replication Samuel Stokes A thesis presented for the degree of Doctor of Philosophy in the College of Medical, Veterinary, and Life Sciences Biotechnology and Biological Sciences Research Council Doctoral Training Partnership programme - University of Glasgow Centre for Virus Research (CVR) S Stokes, 2018 2 Abstract Approximately half the world’s human population is at risk of infection from mosquito-borne arboviruses. Currently, interactions between the mosquito antiviral response and infecting arboviruses remains poorly understood; deciphering these will be crucial to the development of novel methods to limit replication and transmission that could help control future outbreaks. Previous mammalian studies have shown that the Homo sapiens Small Ubiquitin-related Modifier (SUMO) pathway plays a fundamental role in multiple aspects of cell biology, including the regulation of host cell immunity. However, this pathway and its impact on arbovirus replication remain uncharacterised in mosquito hosts such as Aedes aegypti (Ae. aegypti; Aa). Comparison between the Ae. aegypti and H. sapiens (Hs) SUMOylation pathways demonstrated a high degree of amino acid sequence and structural similarity. The most notable predicted difference is the lack of ability of AaSUMO to form poly-SUMO chains, which have important functions in H. sapiens. Biochemical analysis of the AaSUMOylation pathway identified a conserved function, and confirmed that AaSUMO could not efficiently form poly-SUMO chains, unlike HsSUMO3 (its closest H. sapiens homologue) due to the absence of an internal SUMO conjugation motif. Catalytically inactive mutants revealed the necessity of AaPIAS (Protein inhibitor of activated STAT) to induce the formation of poly- SUMO chains. Confocal microscopy confirmed that AaSUMO protein is expressed in haemocytes, the salivary glands, ovaries, and midgut, all of which are sites of arboviral replication. Q-PCR investigations have also revealed the AaSUMOylation pathway to be ubiquitously expressed. In vitro depletion of the AaSUMOylation pathway led to significantly enhanced levels of Zika, Semliki Forest, and Bunyamwera virus replication, identifying a vital role for AaSUMOylation in the suppression of these arboviruses. Subsequent studies in H. sapiens cells have also identified a significant role for HsPIAS1 in suppressing the replication of Zika, Semliki Forest, and Bunyamwera viruses. Furthermore, depletion of HsSUMO1 significantly enhanced the replication of Bunyamwera virus, indicating that SUMOylation suppresses arbovirus in a virus dependent manner. Collectively, these data have identified a novel role for the SUMOylation pathway in S Stokes, 2018 3 suppressing arbovirus replication in both the vertebrate and invertebrate species in which arboviruses replicate. S Stokes, 2018 4 Contents Abstract 2 Contents 4 List of Figures 9 List of Tables 11 Acknowledgements 12 Authors Declaration 15 List of Abbreviations 16 1. Introduction 24 1.1. Overview 25 1.2. Importance of studying vector-borne diseases 26 1.2.1. Aedes aegypti 26 1.2.1.1. Mosquito-arbovirus interactions 29 1.3. Arboviruses 32 1.3.1. Alphaviruses 34 1.3.1.1. Alphavirus epidemiology 35 1.3.1.2. Alphavirus disease 36 1.3.1.3. Alphavirus replication 37 1.3.2. Flaviviruses 40 1.3.2.1. Flavivirus epidemiology 41 1.3.2.2. Flavivirus disease 41 1.3.2.3. Flavivirus replication 43 1.3.3. Orthobunyaviruses 45 1.3.3.1. Orthobunyavirus epidemiology 47 1.3.3.2. Orthobunyavirus disease 47 1.3.3.3. Orthobunyavirus replication 48 1.4. Post-translational modification 51 1.4.1. Ubiquitin 51 1.4.2. Overview of the Small Ubiquitin-like MOdifer (SUMO) protein 53 1.4.2.1. SUMO proteins 54 1.4.2.2. SUMO interaction motifs (SIMs) 57 1.4.2.3. SUMO pathway 57 1.4.2.3.1. Activating enzyme 59 1.4.2.3.2. Conjugating enzyme 59 1.4.2.3.3. Ligating enzyme 60 1.4.2.3.4. Sentrin specific proteases (SENPs) 62 1.5. Interactions between the viruses and the immune responses regulated by SUMO 63 S Stokes, 2018 5 1.5.1. Vertebrate innate immune response 63 1.5.1.1. Alphaviruses and innate immune evasion 67 1.5.1.2. Flaviviruses and innate immune evasion 70 1.5.1.3. Orthobunyaviruses and innate immune evasion 74 1.5.2. Invertebrate innate immune response 76 1.5.2.1. Alphaviruses and Ae. aegypti immune response 80 1.5.2.2. Flaviviruses and Ae. aegypti immune response 81 1.5.2.3. Orthobunyaviruses and Ae. aegypti immune response 82 1.5.3. SUMOylation pathway interactions with viruses 83 1.5.4. SUMO and arboviruses 84 1.6. Premise and primary objectives of the project 85 2. Materials & Methods 86 2.1. Materials 87 2.1.1. Cells and cell culture reagents 87 2.1.1.1. Cell lines utilised 87 2.1.1.2. Cell culture reagents utilised 89 2.1.1.3. Viruses 90 2.1.2. Antibodies 92 2.1.3. Plasmids 94 2.1.4. Primer sequences 98 2.1.5. Reagents, chemicals, buffers, and kits 101 2.1.5.1. Commercial kits 101 2.1.5.2. Buffers made in house 102 2.1.5.3. Reagents used 103 2.1.6. Enzymes 105 2.1.7. Reagents used for prokaryotic work 106 2.1.7.1. Bacterial strains and culture media 106 2.1.6.2. Buffers for protein purification from bacterial extracts 106 2.2. Methods 108 2.2.1. Cloning and DNA manipulation 108 2.2.1.1. DNA quantitation 108 2.2.1.2. Polymerase Chain Reaction (PCR) amplification 108 2.2.1.3. Restriction endonuclease digestion 110 2.2.1.4. Agarose gel electrophoresis 110 2.2.1.5. DNA Ligation 110 2.2.1.6. Transforming competent bacteria 111 2.2.1.7. Growing bacterial cultures of E. coli DH5α 111 2.2.1.8. Isolation and amplification of DNA using kits 112 2.2.1.9. Sequencing by commercial companies 112 2.2.1.10. Site directed mutagenesis of plasmid DNA by PCR 113 S Stokes, 2018 6 2.2.1.11. dsRNA synthesis for cell culture 115 2.2.1.12. dsRNA synthesis for adult Ae. aegypti 115 2.2.2. Purification of recombinant proteins 119 2.2.2.1. Expression in recombinant BL21 (DE3) 119 2.2.2.2. Purification of 6xHis tagged proteins 119 2.2.2.3. Purification of Strep.II tagged proteins 120 2.2.2.4. Quantification of proteins by BSA titration 120 2.2.3. In vitro assays of recombinant proteins 121 2.2.3.1. SUMOylation assays 121 2.2.4. Cell culture methods 122 2.2.4.1. Maintenance, growth, and passaging of cells 122 2.2.4.2. Seeding of cells 122 2.2.4.3. Transfection of mammalian cells 123 2.2.4.4. Lentivirus transduction of cells 124 2.2.4.5. dsRNA transfection of mosquito cells 124 2.2.5. Virology 125 2.2.5.1. Propagation of SFV 125 2.2.5.2. Propagation of BUNV 125 2.2.5.3. Propagation of ZIKV 125 2.2.5.4. Determining viral titre 126 2.2.5.5. Infection of cultured cells 127 2.2.6. Analytical techniques 128 2.2.6.1. Sodium dodecyl sulfate – Polyacrylamide gel electrophoresis 128 2.2.6.2. Coomassie staining of SDS-PAGE gels 128 2.2.6.3. Western blotting 128 2.2.6.4. RNA extraction 129 2.2.6.5. Complementary DNA (cDNA) synthesis 129 2.2.6.6. Quantitative polymerase chain reaction (q-PCR) 130 2.2.6.7. ICC-staining plaque assay 131 2.2.6.8. In Cell Western Blot (ICWB) 131 2.2.7. Reporter assays 133 2.2.7.1. Firefly-luciferase reporter assay 133 2.2.7.2. NanoLuciferase reporter assay 133 2.2.7.3. Protein quantification assay 134 2.2.8. Bioinformatics 135 2.2.8.1. Joined Advanced SUMOylation site and SIM Analyser (JASSA) 135 2.2.8.2. Modelling software 135 2.2.8.3. Amino acid alignment 136 2.2.8.4. Statistical analysis 136 2.2.9. Cloning strategies used in this study 137 S Stokes, 2018 7 2.2.9.1. Cloning of pACYC-AaSAE1/2 137 2.2.9.2. Cloning of pET28a-SUMO chimaera and pET28a-SUMO chimaera K11R 139 2.2.9.3. Cloning of pET45b-AaUbc9 and pET45b-AaUbc9 C93S 140 2.2.9.4. Cloning of pET45b-AaPIAS and pET45-AaPIAS C371A 141 2.2.9.5. Cloning of pET28a-AaSUMO 141 3. Conservation of the SUMOylation pathway between Ae. aegypti and H. sapiens 142 3.1. Overview 143 3.2. Conservation of the SUMOylation pathway 145 3.2.1. Conservation of SUMO 145 3.2.2. Conservation of Ubc9 148 3.2.3. Conservation of SAE1/2 151 3.2.4. Conservation of PIAS 154 3.2.5. Confirmation of pathway expression 158 3.2.6. Summary 164 3.3.