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An Investigation of the Mechanisms Underlying HIV-1-Mediated Inflammasome Activation

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An Investigation of the Mechanisms Underlying HIV-1-Mediated Inflammasome Activation An investigation of the mechanisms underlying HIV-1-mediated inflammasome activation Christopher JK Ward Doctorate of Philosophy in Medicine Supervised by Dr. Martha Triantafilou & Prof. Kathy Triantafilou Submitted July 2017 i DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed ……………………………………………………… (candidate) Date 01.07.2017 STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of Doctorate of Philosophy (Medicine) Signed ………………………………………….…………… (candidate) Date 01.07.2017 STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated, and the thesis has not been edited by a third party beyond what is permitted by Cardiff University’s Policy on the Use of Third Party Editors by Research Degree Students. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ……………………………………….……….…… (candidate) Date 01.07.2017 STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ……………………………………………..…..….. (candidate) Date 01.07.2017 ii Table of Contents Table of Contents ............................................................................................................ iii Acknowledgements ........................................................................................................ vii Dedication ..................................................................................................................... viii Summary ......................................................................................................................... ix Glossary of Abbreviations ................................................................................................. x Chapter 1: Introduction ....................................................................................................1 1. Pattern recognition receptors: first line of defence against infection ................................... 2 1.1 Toll-like receptors: the first pattern recognition receptor family to be discovered ................ 2 1.2 Toll-like receptor signalling pathways ....................................................................................15 1.3 Structure and function of the NLR inflammasome .................................................................20 1.4 Different NLRs recognise different pathogens .......................................................................24 1.5 Unknowns in PRR sensing .......................................................................................................36 2. HIV-1 and Acquired Immune Deficiency Syndrome (AIDS) ............................................. 37 2.1 The natural history of HIV-1 infection ....................................................................................37 2.2 Virology and lifecycle of HIV-1 ................................................................................................40 2.3 Treatments for HIV-1 infection ...............................................................................................53 3. HIV-1-pattern recognition receptor interactions ............................................................... 59 3.1 HIV-1 interacts with TLRs ........................................................................................................59 3.2 HIV-1 interacts with NLRs .......................................................................................................61 4.0 Viroporins interact with the innate immune system ........................................................ 64 4.1 Structure and function of viroporins ......................................................................................64 iii 4.2 Viroporins activate the NLRP inflammasome .........................................................................65 4.3 HIV-1 Vpu viroporin.................................................................................................................68 5. Aims .................................................................................................................................. 71 Chapter 2: Methods & Materials ..................................................................................... 72 1. Tissue Culture ................................................................................................................... 73 1.1 Cell Types ................................................................................................................................73 1.2 Isolation of human peripheral blood mononuclear cells ........................................................74 1.3 Freezing and thawing cells ......................................................................................................74 1.4 Cell counting & viability ..........................................................................................................74 2 Antibodies, markers, reagents ............................................................................................ 76 2.1 Antibodies & markers .............................................................................................................76 2.2 Standard laboratory chemicals ...............................................................................................79 2.3 Inhibitors .................................................................................................................................80 3 Plasmids ............................................................................................................................. 81 3.1 Transformation .......................................................................................................................81 3.2 Isolation and electrophoresis..................................................................................................82 4. HIV-1 Reagents & Protocols ............................................................................................. 88 4.1 HIV-1 envelope glycoproteins .................................................................................................88 4.2 HIV-1 Protein Plasmids ............................................................................................................88 4.3 Virus production and infectivity assay ....................................................................................90 4.4 Limulus amoebocyte lysate assay ...........................................................................................92 7. Flow cytometry ................................................................................................................. 93 7.1 Principles ......................................................................................................................... 93 7.2 Analysing PRR expression via flow cytometry ........................................................................95 8. Western Blotting ............................................................................................................... 96 8.1 Principles .................................................................................................................................96 iv 8.2 Detection of pattern recognition receptor activation ............................................................97 9 Cytokine analysis ............................................................................................................... 99 9.1 Principles .................................................................................................................................99 Figure 35. Mechanism of cytokine detection with reporter cells (Image adapted from Invivogen). ..................................................................................................................... 99 9.2 Detection of specific cytokines using reporter cells .............................................................100 9.3 Detection of specific cytokines using enzyme-linked immunosorbance assay ....................100 10. Confocal microscopy ..................................................................................................... 102 10.1 Principles .............................................................................................................................102 11. Förster Resonance energy transfer (FRET) ....................................................................... 104 13. Statistics & Software ..................................................................................................... 106 Chapter 3: HIV-1 envelope glycoprotein triggers an innate immune response ................ 108 1.1 Introduction ............................................................................................................ 109 1.2 HIV-1 envelope glycoprotein gp120 activates the innate immune system......................... 110 1.3 HIV-1 gp120 upregulates TLRs and NLRs .......................................................................... 111 1.4 HIV-1 gp120 upregulates TLRs and NLRs in MOLT4 T cells ................................................ 125 1.5 HIV-1 gp120 inflammasome activation is NLRP3 dependent............................................
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