Flaviviruses Versus the Host Cell, and Evolution in the Primate Interferon Response by Alison Ruth Gilchrist B.Sc., University of California at San Diego Athesissubmittedtothe Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Cellular and Developmental Biology 2020 Committee Members: Sara L. Sawyer, Chair Robert L. Garcea Rushika Perera Robin D. Dowell Sabrina L. Spencer ii Gilchrist, Alison Ruth (Ph.D., Molecular Cellular and Developmental Biology) Flaviviruses Versus the Host Cell, and Evolution in the Primate Interferon Response Thesis directed by Prof. Sara L. Sawyer Long-term interactions between viruses and their hosts often develop into genetic arms races, which result in fast-evolving proteins (i.e. proteins evolving under positive natural selection), especially in immune proteins. A bioinformatic screen of proteins in a component of the primate innate immune response, the interferon system, demonstrated that proteins farther downstream of interferon induction are more likely to be evolving under positive natural selection compared to proteins in interferon induction pathways. One of the proteins under positive selection in this screen, STING, is a known target of proteases from a group of viruses called flaviviruses. The human haplotypes of STING (three of which are studied in this work) demonstrate a range of phenotypes of antagonism and interferon induction that may help explain the evolutionary history of this crucial immune protein. The cleavage of STING demonstrates that the dengue virus protease targets host proteins for cleavage as well as viral proteins. In an attempt to identify novel targets of the dengue virus protease, a machine learning screen was used to predict possible motifs based on known motifs. This resulted in the identification of DGAT2, a newly described target of flavivirus proteases. The ability to cleave DGAT2, a host protein involved in maintaining lipid homeostasis, improves dengue virus replication, and is a conserved property of all flavivirus proteases tested. DGAT2 is not evolving under positive selection, making it a host-virus antagonistic interaction that has not resulted in an evolutionary arms race. However, identifying and describing this host-virus interaction helps us understand how dengue virus and other flaviviruses alter the host lipid environment during replication. Dedication For my parents. ”To know how much there is to know is the beginning of learning to live.” -Dorothy West (The Richer, the Poorer) iv Acknowledgements To Sara: thank you for setting up the support systems that propped me and my science up when we needed it, and for caring about the lab safety and organization that made being a bench scientist so fun. And thank you to Bob, Rushika, Robin, Sabrina, and one-time committee member David for being unfailingly kind, supportive, and helpful. Thank you to Nicholas Meyerson for being an unwavering hero. You helped me so much, for so long, that I will understand if you never want to speak to me again (but please do). Alex Stabell and Maryska Kaczmarek: thank you so much for welcoming me in to the lab, and for your help and friendship over the years. Thank you to Vanessa Bauer for being an incredible lab manager; I will never forgive you for setting an impossible standard. Thank you to Elena Judd for being a wonderful technician, and then an even more wonderful undergraduate researcher—you’re going to do great things in the future. Everyone else I met and worked with in the Sawyer Lab, including but not limited to: Cody Warren, Qing Yang, Camille Paige, Kyle Clark, Will Fattor, Arturo Barbachano-Guerrero, Joe Timpona, Emma Worden-Sapper, Sharon Wu, Obaiah Dirasantha: I’ll never forget any of you! And I can’t wait to check in on everyone over the coming years. Thanks to my mom for letting me cry over the phone so many times, and also for bragging about me so much, making it extremely hard to drop out of grad school. Thanks to my dad for helping me climb mountains, physical and metaphorical, my whole life. Thanks to Andrew and Daniel for nothing related to research, but everything related to sibling solidarity, and to Lena Meyer for being my not-a-sister sister, and an endless well of support and love when I needed it. Thank you to MCDB for a wonderful six years. To the entering MCDB class of 2014: I can’t v believe I was so lucky to spend my PhD with such wonderful classmates. To Adrian, Graycen, Julie, Daniel, Brad, Kate, and Abby: an extra special thank you for being part of what kept me sane. For every Science Bu↵, Nerd-Niter, and ComSciCon-RMW committee member I spent time with in the last six years: I hope I learned even a small portion of what you all have to teach scientists. To every other friend and colleague that I don’t have the space to thank: know that I appreciated you dearly and you helped get me through grad school. vi Contents Chapter 1 Introduction 1 1.1 Viruses and hosts in evolutionary combat . 1 1.2 Positiveselectionintheinterferonpathway . 4 1.3 STING: an immune protein that is a target of flavivirus proteases . 5 1.4 Using machine learning to predict new targets of flavivirus proteases . 6 1.5 DGAT2: a novel target of flavivirus proteases . 7 1.6 Thesis organization . 9 2 Positive selection in the interferon pathway 10 2.1 Positive natural selection . 10 2.2 Positiveselectioninimmunepathways . 11 2.3 Theinterferonresponse .................................. 12 2.4 Screening for positive selection in interferon pathways . 14 2.5 Characterization of multiple sequence alignments . 17 2.6 Interferon-stimulated genes experience more intense positive selection than interferon- induction genes or randomly-selected genes . 19 2.7 Discussion.......................................... 24 2.8 Methods........................................... 25 vii 3 STING: an immune protein that is a target of flavivirus proteases 30 3.1 Flaviviruses, and dengue viruses in particular, are world-wide pathogens with major consequences for human health . 30 3.2 Human STING and the interferon response . 32 3.3 Human STING, but not human 78Q STING, is cleaved by multiple flaviviruses . 33 3.4 RodentSTINGisunderpositiveselection . 41 3.5 The interferon response is stimulated by STING transfection . 42 3.6 Active dengue virus protease inhibits interferon production . 44 3.7 Small di↵erences in virus replication in cell lines expressing di↵erent STING alleles . 47 3.8 Not all flavivirus proteases inhibit the STING-dependent interferon response . 49 3.9 Discussion.......................................... 49 3.10Methods........................................... 53 4 DGAT2: a novel target of flavivirus proteases identified by machine learning 57 4.1 Predicting targets of the dengue virus protease by machine learning . 57 4.2 DGATiscleavedbythedenguevirusprotease . 63 4.3 Mutation of the DGAT2 cleavage motif reduces viral infection. 65 4.4 Confirmation with DGAT2 KO A549s . 73 4.5 ChemicallyinhibitingDGAT2activity . 74 4.6 Cleavage of DGAT2 is conserved in the flavivirus family. 77 4.7 The cleavage of DGAT2: discussion . 79 4.8 Attempted further validation of the machine learning approach to predicting targets offlavivirusproteases ................................... 81 4.9 Methods........................................... 92 5 Conclusion 100 viii Bibliography 102 Appendix A Genes Analyzed in the Interferon Positive Selection Screen 113 B Host Proteins Predicted by Machine Learning 126 ix Tables Table 2.1 Many interferon genes are known to be evolving under positive selection. 13 2.2 Genes in the interferon induction pathway and genes stimulated by interferon evolv- ingunderpositiveselection. .. 26 x Figures Figure 1.1 An example of the arms race between virus and host proteins . 3 1.2 DGAT2Biochemistry ................................... 8 2.1 Simplified diagram of the interferon response. 16 2.2 Quality and equity metrics for the three families of multiple sequence alignments compared........................................... 18 2.3 Characterization of multiple species alignments . 21 2.4 Interferon-stimulated genes have a higher whole-gene dN/dS value than other genes, and have more codons under positive selection than other genes. 23 3.1 DengueviruscleavesSTINGduringinfection . 34 3.2 The SNPs of HAQ STING are geographically distinct . 36 3.3 The 78Q SNP prevents cleavage of human STING . 38 3.4 DENV2 cleaves STING and HAQ STING, but not 78Q STING . 39 3.5 Most flaviviruses cleave STING and HAQ STING, but not 78Q STING . 40 3.6 Rodent Sting1 is under positive selection, but only in the Hystricomorpha clade. 43 3.7 Transfecting STING induces interferon production . 45 3.8 Transfecting STING and DENV2 protease dampens interferon production. 46 3.9 STING SNPs and protease antagonism. 48 3.10 Non-cleavable STING reduces DENV2 replication in A549s. 50 xi 3.11 STING alleles and flavivirus protease antagonism. 51 3.12 STING alleles and flavivirus protease antagonism, part 2. 52 4.1 The dengue virus polyprotein is cleaved by the dengue virus protease. 60 4.2 Schematic of the machine learning protocol. 62 4.3 DGAT2 motif identified and the structure of DGAT2. 64 4.4 DGAT2iscleavedbythedenguevirusprotease. 66 4.5 DGAT2 cleavage product not significantly degraded by the proteasome . 67 4.6 Wild-type A549 cells complemented with mutant DGAT2 did not inhibit dengue virusreplication. ...................................... 69 4.7 Endogenous DGAT2 is significantly reduced after transfection of DGAT2-targeting siRNA............................................. 70 4.8 Non-cleavable DGAT2 reduces DENV2 replication. 72 4.9 The presence of non-cleavable DGAT2 inhibits dengue replication in DGAT2 knock- outcells............................................ 75 4.10 DGAT2 inhibition in DGAT2 mutant cell lines did not result in significantly higher denguevirusreplication................................... 76 4.11 DGAT2 cleaved by all tested flavivirus proteases . 78 4.12 Proposed model for flavivirus cleavage of DGAT2 . 80 4.13 Example screening of predicted host proteins by western blotting. 83 4.14 The vast majority of predicted proteins were not cleaved by the dengue virus protease.