TARGETING NEGATIVE REGULATORS OF IMMUNE SIGNALING PATHWAYS TO DISRUPT THE HIV-1 LIFE CYCLE By JARED PAUL TAYLOR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Jared Paul Taylor To the giants who came before us, on whose shoulders we stand ACKNOWLEDGMENTS I would like to thank my mentor Mark Wallet for giving me the opportunity to pursue my graduate research in his lab. He has been a wonderful mentor over the years and I have learned so much from him. I also would like to thank Maureen Goodenow, Stephanie Karst, Naohiro Terada, and Edward Scott for serving on my advisory committee and for their support and advice. I would also like to thank all the Wallet lab members past and present who have been coworkers as well as friends for making the work environment enjoyable and collaborative. I would also like to thank Dave Bloom for sponsoring me on the Infectious Disease T32 training grant for three years. This afforded me many unique opportunities with funding for not only my graduate stipend and tuition, but also for supplies and traveling to conferences greatly enriching my graduate experience. I want to also express my appreciation for all of the administrative staff that have helped with so many things through the years. The IDP office staff have been very helpful especially in the beginning of the program. I especially want to thank Karen Cox in the Pathology office for all of the many things she does for our lab. I also thank Kris Minkoff and Debbie Burgess in the Microbiology and Molecular Genetics office for their support with things related to the Immunology and Microbiology concentration and the training grant. I would also like to thank my family for their support throughout the years. I also thank my friends here in Gainesville who have made getting through the program much easier. Finally, I would like to thank my dog Winston who has been my constant loyal companion as I have gone through graduate school. 4 TABLE OF CONTENTS Page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 12 CHAPTER 1 BACKGROUND ...................................................................................................... 14 Discovery of HIV/AIDS ............................................................................................ 14 Natural History of HIV/AIDS .................................................................................... 15 Where Do We Go from Here? ................................................................................. 16 HIV-1 Lifecycle ........................................................................................................ 17 Outcomes of the Life Cycle ..................................................................................... 23 Immune Control of HIV-1 ........................................................................................ 37 2 DEPLETION OF USP18 ENHANCES TYPE I IFN RESPONSIVENESS AND RESTRICTS HIV-1 INFECTION IN MACROPHAGES ........................................... 46 Introduction ............................................................................................................. 46 Methods and Materials............................................................................................ 54 Results .................................................................................................................... 64 Discussion .............................................................................................................. 71 Figures .................................................................................................................... 78 Tables ..................................................................................................................... 98 3 HARMINE BOOSTS REACTIVATION OF LATENT HIV-1 BY LATENCY REVERSING AGENTS ......................................................................................... 100 Introduction ........................................................................................................... 100 Methods and Materials.......................................................................................... 105 Results .................................................................................................................. 108 Discussion ............................................................................................................ 112 Figures .................................................................................................................. 116 Tables ................................................................................................................... 130 4 CONCLUDING REMARKS ................................................................................... 131 5 LIST OF REFERENCES ............................................................................................. 136 BIOGRAPHICAL SKETCH .......................................................................................... 192 6 LIST OF TABLES Table Page 2-1 Primer Sequences .............................................................................................. 98 2-2 HIV-1-Induced Gene Expression ........................................................................ 99 3-1 PMA-Induced Gene Expression with Harmine.................................................. 130 7 LIST OF FIGURES Figure Page 2-1 Type I IFN restricts HIV-1 replication in TZM-bl cells .......................................... 78 2-2 IFNAR blocking inhibits HIV-1 replication ........................................................... 79 2-3 HIV-1 induces an IFN-like response in MDMs .................................................... 80 2-4 USP18 expression is induced by HIV-1 and is dependent on type I IFN signaling ............................................................................................................. 81 2-5 USP18 knockdown by siRNA makes THP-1 cells and MDMs, but not TZM-bl cells, refractory to HIV-1 infection ....................................................................... 82 2-6 USP18 knockdown by shRNA in THP-1 cells ..................................................... 84 2-7 shRNA knockdown of USP18 inhibits HIV-1 replication in THP-1 cells and enhances IFN-β-induced STAT activation .......................................................... 85 2-8 siRNA knockdown of USP18 enhances STAT activation and modulates the transcriptome in IFN-β-treated MDMs ............................................................... 87 2-9 ISG expression is enhanced in USP18 deficient THP-1 Cells and MDMs .......... 89 2-10 Workflow for generating iPSC-derived monocytes ............................................. 91 2-11 Characterization of iMacs ................................................................................... 92 2-12 iMacs support HIV-1 replication .......................................................................... 93 2-13 CRISPR/Cas9 knockout of USP18 in iPSCs ...................................................... 94 2-14 USP18-/- iMacs have enhanced STAT phosphorylation ...................................... 95 2-15 Knockout of USP18 restricts HIV-1 replication in iMacs ..................................... 96 2-16 USP18 tunes the IFN response to allow for efficient HIV-1 replication ............... 97 3-1 Chemical structure of DYRK1A inhibitors ......................................................... 116 3-2 DYRK1A inhibitors boost LRA-induced HIV-1 reactivation in 5A8 cells ............ 117 3-3 Harmine boosts clinically-relevant LRA-induced HIV-1 reactivation in 5A8 cells .................................................................................................................. 118 3-4 Harmine boosts the MFI of GFP induced by LRAs ........................................... 119 8 3-5 Harmine boosts bryostatin-induced gag expression in J-Lat cells .................... 120 3-6 Harmine boosts LRA-induced gag expression in J-Lat cells ............................. 121 3-7 Harmine boosts the magnitude of SAHA reactivation, but not the frequency of reactivated cells ................................................................................................ 122 3-8 DYRK1A CRISPR knockout in 5A8 cells .......................................................... 123 3-9 DYRK1A knockout in 5A8 cells does not boost PMA-induced reactivation of latent HIV-1....................................................................................................... 124 3-10 Harmine boosts PMA-induced phosphor-ERK1/2 expression .......................... 125 3-11 MEK/ERK inhibitor abrogates effect of harmine in PMA treated 5A8 cells ....... 126 3-12 Jurkat T cells that were transduced with lentivirus NFκB or NFAT luciferase reporters or a negative control lentivirus were treated with different doses of PKC overnight .................................................................................................