TEMPERATURE-DEPENDENT EFFICACY OF THE IFN-α/β RESPONSE AGAINST CHIKUNGUNYA AND OTHER ARBOVIRUSES by Whitney Christine Lane B.S. in Biology, Purdue University-Fort Wayne, 2012 Submitted to the Graduate Faculty of The School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Virology and Microbiology University of Pittsburgh 2018 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Whitney Lane It was defended on July 10, 2018 and approved by William Klimstra, PhD, Associate Professor of Immunology Neal DeLuca, PhD, Professor of Microbiology and Molecular Genetics Robert Binder, PhD, Associate Professor of Immunology Amy Hartman, PhD, Assistant Professor of Infectious Diseases and Microbiology John Williams, MD, Professor of Pediatrics Doug Reed, PhD, Associate Professor of Immunology Dissertation Advisor: William Klimstra, PhD, Associate Professor of Immunology ii Copyright © by Whitney Lane 2018 iii TEMPERATURE-DEPENDENT EFFICACY OF THE IFN-α/β RESPONSE AGAINST CHIKUNGUNYA AND OTHER ARBOVIRUSES Whitney Christine Lane University of Pittsburgh, 2018 Chikungunya virus (CHIKV) is a mosquito-transmitted arbovirus in the genus Alphavirus that has reemerged in recent years to cause explosive epidemics of acute and chronic arthritic disease across the globe. Like other arboviruses, CHIKV has evolved to replicate efficiently in both its arthropod vector and its vertebrate hosts, which represent two very different thermal environments. Arthritogenic alphaviruses such as CHIKV preferentially replicate and cause disease in distal small joints that are lower in temperature than the body core. The type-I interferon (IFN-α/β) system is a critical innate immune response for controlling arbovirus infection. Recent evidence suggests that IFN-α/β is less effective in controlling virus infection at temperatures below the body core temperature. Therefore, we hypothesized that arboviruses such as CHIKV may exploit a weakened innate immune environment in low-temperature joint tissues to fuel exacerbated viral replication and disease at these sites. To address this hypothesis, we examined the efficacy of IFN-α/β induction and antiviral efficacy against CHIKV and other arboviruses at different temperatures within the normal physiological range. In vitro, we found that mild hypothermia significantly compromised both the induction and effector arms of the IFN-α/β response compared to normal or febrile-range temperatures against diverse arboviruses. We narrowed this effect to delayed and reduced transcription of effector genes, as upstream signaling pathways were not attenuated by hypothermia, and demonstrated differential temperature-sensitivity profiles of interferon- stimulated gene (ISG) induction versus some lipopolysaccharide (LPS)-inducible genes. Finally, iv mice with reduced core temperatures suffered exacerbated CHIKV replication and disease compared with normal mice during the acute phase of infection, and this phenotype was dependent upon a functional IFN-α/β system. Reduced core temperature during the first few days of CHIKV infection also resulted in higher persistent viral signal at chronic time points, and temperature reduction >30 days post infection resulted in significantly increased viral protein production at persistently-infected sites. Together, these data indicate that reduced tissue temperature results in attenuated IFN-α/β and ISG gene transcription, leaving these tissues more vulnerable to arboviral infection than warmer sites. CHIKV can take advantage of this dynamic to infect and persist in low-temperature tissues in vivo. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XIV 1.0 INTRODUCTION ........................................................................................................ 1 1.1 ARBOVIRUSES .................................................................................................. 1 1.1.1 Major characteristics of arbovirus genera and associated diseases ............ 3 1.1.1.1 Genus Alphavirus................................................................................... 3 1.1.1.2 Genus Flavivirus .................................................................................... 5 1.1.1.3 Order Bunyavirales............................................................................... 6 1.1.1.4 Family Reoviridae .................................................................................. 8 1.1.2 Common features of arbovirus transmission and ecology ........................... 9 1.1.3 Features of arbovirus pathogenesis and immune response in humans .... 11 1.2 CHIKUNGUNYA VIRUS ................................................................................. 14 1.2.1 Cellular replication ........................................................................................ 15 1.2.2 Distribution and ecology ............................................................................... 17 1.2.3 Pathogenesis and disease in humans ............................................................ 20 1.2.4 Adaptive immune response ........................................................................... 27 1.2.4.1 T cell response ..................................................................................... 27 1.2.4.2 B cell response ..................................................................................... 29 1.2.5 Animal models of CHIKV infection ............................................................. 31 vi 1.2.5.1 Neonatal and immunocompromised murine lethal challenge models 31 1.2.5.2 Adult wild-type murine acute arthritic models ................................ 31 1.2.5.3 Mouse models of chronic CHIKV infection ...................................... 32 1.2.5.4 Nonhuman primate models ................................................................ 33 1.3 TYPE-I INTERFERON .................................................................................... 33 1.3.1 Definition and classification .......................................................................... 35 1.3.2 Induction pathways ....................................................................................... 36 1.3.2.1 Toll-like receptor signaling ................................................................ 36 1.3.2.2 RIG-I-like receptor signaling ............................................................. 38 1.3.2.3 IFN gene transcription........................................................................ 40 1.3.3 Effector pathways .......................................................................................... 41 1.3.3.1 Canonical JAK-STAT signaling ........................................................ 42 1.3.3.2 Accessory signaling pathways ............................................................ 42 1.3.3.3 ISG transcriptional regulation ........................................................... 44 1.3.4 ISG functions and the antiviral state ........................................................... 46 1.3.4.1 Myxovirus resistance (Mx) ................................................................. 48 1.3.4.2 Interferon-induced protein with tetracopeptide repeats (IFIT) ..... 48 1.3.4.3 Interferon-inducible transmembrane (IFITM) ................................ 49 1.3.4.4 Zinc finger antiviral protein (ZAP) ................................................... 50 1.3.4.5 Interferon-stimulated gene 15 kDa (ISG15) ..................................... 50 1.3.4.6 Interferon-stimulated gene 20 kDa (ISG20) ..................................... 50 vii 1.3.4.7 Virus inhibitory protein, endoplasmic reticulum-associated, IFN- inducible (Viperin) ............................................................................................. 51 1.3.5 Effects of IFN-α/β on innate and adaptive immunity ................................. 52 1.4 PHYSIOLOGICAL TEMPERATURE VARIATION ................................... 54 1.4.1 Mechanisms of thermoregulation for core temperature maintenance ..... 55 1.4.1.1 Physiological and behavioral responses to cold exposure ............... 56 1.4.2 Physiological and behavioral responses to heat exposure .......................... 60 1.4.3 Temperature variation between anatomical compartments ..................... 61 1.4.4 Physiological factors affecting human body temperature ......................... 64 1.4.4.1 Impaired circulation ........................................................................... 64 1.4.4.2 Fever ..................................................................................................... 65 1.4.5 Intracellular responses to temperature change .......................................... 66 1.4.5.1 Heat shock response ............................................................................ 67 1.4.5.2 Cold shock response ............................................................................ 70 1.4.6 Effects of physiological temperature variation on immune responses ..... 74 1.4.6.1 Fever/hyperthermia ............................................................................ 74 1.4.6.2 Hypothermia ........................................................................................ 77 1.5 HYPOTHESIS ................................................................................................... 78 2.0 EFFICACY OF THE INTERFERON-Α/Β RESPONSE VERSUS ARBOVIRUSES IS TEMPERATURE-DEPENDENT ..........................................................