Molecular Mechanisms Governing Host Responses and Cellular Tropism During Flavivirus Infection Pubblico
Quicke, Kendra (Summer 2018)
Abstract
The RIG-I-like receptors (RLRs) are crucial to initiating innate immune responses to multiple flaviviruses. LGP2, a non-signaling member of this family, is a central regulator of the RIG-I and MDA5 pathways. However, the molecular mechanism underlying the regulatory function of LGP2 is still unclear. Additionally, very little is currently known about the specific role of RLRs in responding to Zika virus (ZIKV), a re-emerging flavivirus. ZIKV has exhibited several characteristics that are not observed with other flaviviruses, and as such it should not be assumed that the same host factors will efficiently control infection with this virus. Antiviral responses also vary based on cell type. As ZIKV appears to have a broad tropism, including innate immune cells, neuroprogenitor cells, and placental cells, it is important to evaluate the responses induced in each of these tissues. The work presented within this dissertation provides insight into the molecular mechanisms of RLR signaling regulation, antiviral responses to ZIKV infection, and ZIKV cellular tropism. Specifically, we demonstrate a heretofore undescribed mechanism of LGP2 negative regulation, wherein LGP2 inhibits early RIG-I activation by preventing K63-ubiquitination of the RIG-I CARDs through an interaction with the E3 ligase TRIM25. We also explore the susceptibility and immune responses to ZIKV infection within human primary dendritic cells (DCs), placental macrophages (Hofbauer cells [HCs]), and cytotrophoblasts (CTBs). DCs and HCs, and CTBs to a lesser extent, were found to support productive ZIKV infection. ZIKV induced antiviral gene expression in these cells, but this did not always correspond to increased protein production, particularly in the case of type I interferons (IFNs). Furthermore, ZIKV was found to potently inhibit type I IFN signaling by inhibiting activation of the transcription factors STAT1/2. In addition, the presence of cross-reactive flavivirus antibodies may further impact the immune responses and tropism of ZIKV. DENV-induced monoclonal antibodies cross-reacted with ZIKV and resulted in attenuated antiviral responses in HCs and enhanced infection of HCs and human placental explant tissues, suggesting a mechanism for the vertical transmission of ZIKV. These findings may have translational consequences on the development of new vaccines and antiviral therapies.
Table of Contents
Chapter 1: Introduction 1
Introduction to RIG-I-like receptors 2
Structure of the RLRs 2
Function of RIG-I and MDA5 6
Function of LGP2 8
Introduction to Zika virus 14
Flavivirus innate immunology 16
Potential impact of cross-reactive flavivirus antibodies 23
Biology of the placenta 26
RLRs and ZIKV infection 30
Chapter 2: The RNA Helicase LGP2 Negatively Regulates RIG-I Signaling by Preventing TRIM25-mediated CARD Ubiquitination 31
Introduction 32
Methods 34
Results 39
Discussion 46
Chapter 3: Zika Virus Antagonizes Type I Interferon Responses During Infection of Human Dendritic Cells 60
Introduction 61
Methods 63
Results 69
Discussion 83
Chapter 4: Zika Virus Infects Human Placental Macrophages 109
Introduction 110
Methods 111
Results 117
Discussion 122
Chapter 5: Human Antibody Responses After Dengue Virus Infection are Highly Cross-Reactive to Zika Virus 134
Introduction 136
Methods 138
Results 141
Discussion 146
Chapter 6: Cross-reactive dengue virus antibodies augment Zika virus infection of human placental macrophages 158
Introduction 159
Methods 161
Results 169
Discussion 177
Chapter 7: Discussion 194
Summary of LGP2 study findings 195
Potential factors influencing disparate results 195
The conundrum of LGP2 function during WNV infection 200
LGP2 conclusion: RLR agonists as vaccine adjuvants 203
Summary of ZIKV study findings 204
Further advances in ZIKV tropism and immunology 205
Vertical transmission of other flaviviruses 209
Unanswered questions 212
ZIKV conclusion: A lingering threat 215
Concluding thoughts 216
Works Cited 218
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