Molecular Mechanisms Governing Host Responses and Cellular Tropism During Flavivirus Infection Open Access

Quicke, Kendra (Summer 2018)

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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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