Mechanisms of Flavivirus Antagonism of Innate Immune Signaling in Human Dendritic Cells Open Access

Abstract

West Nile virus (WNV) is a neurotropic flavivirus that remains a leading cause of mosquito-borne encephalitis in the United States. Zika virus (ZIKV), which is closely related to WNV, is an emerging mosquito-borne flavivirus that has sparked a global public health crisis due to a causal linkage to severe neonatal birth defects. Previous work has suggested that dendritic cells (DCs) are important cellular targets during infection with related flaviviruses, including dengue, yellow fever, and Japanese encephalitis viruses. However, the contributions of human DCs during WNV or ZIKV infection remains poorly understood. Here, we utilized primary human cells to demonstrate that monocyte-derived DCs (moDCs) support productive viral replication following infection with WNV and ZIKV. Using a systems biology approach, STAT5 was identified as a regulator of DC activation that was not activated during WNV or ZIKV infection. Consequently, molecules involved in antigen presentation and T cell activation were minimally induced during WNV and ZIKV infection, and functionally, WNV-infected moDCs dampened allogeneic T cell proliferation. Mechanistically, WNV and ZIKV blocked tyrosine phosphorylation of STAT5, and to a lesser extent STAT1 and STAT2, through impairment of Tyk2 and JAK1 activation. ZIKV, but not WNV, also selectively blocked type I IFN protein translation, without affecting the up-regulation of other antiviral proteins. Combined, our studies use primary human cells to reveal novel mechanisms used by WNV and ZIKV to subvert DC activation during productive infection within human moDCs.

The mechanisms and cell types involved in transplacental transmission of ZIKV are poorly understood. Here, we utilized primary human cells isolated from villous tissue of full-term placentae to demonstrate that ZIKV productively replicates within primary human placental macrophages, known as Hofbauer cells (HCs). ZIKV also infected cytotrophoblasts, although viral replication was delayed and more limited. ZIKV infection of HCs promoted up-regulation of T cell co-stimulatory molecules, production of pro-inflammatory cytokines, type I IFN secretion, and strong antiviral gene expression. Combined, our findings support a mechanism of transplacental transmission where ZIKV gains access to the developing fetus by directly infecting placental cells and disrupting the placental barrier.

Table of Contents

TABLE OF CONTENTS Distribution agreement

i

Approval sheet

ii

Abstract cover

iii

Abstract

iv

Cover page

v

Acknowledgements

vi

Table of Contents

vii

List of Figures and Tables

x

Abbreviations

xii

Chapter 1: Introduction

1

Part 1. Neurotropic flaviviruses 1

A) West Nile virus

1 a) WNV disease in humans 2 b) WNV pathogenesis 3 c) WNV immunity 4

B) Zika virus

5

a) ZIKV is causally linked to birth defects during congenital infection

7 b) Transplacental transmission of ZIKV 8

c) ZIKV targets neuroprogenitor cells in the fetal brain

10 d) ZIKV immunity 11

Part 2. Innate immune signaling during WNV and ZIKV infection

14

A) RIG-I like receptor signaling

15

a) MAVS is critical for host control of WNV infection

b) Non-redundant roles of RIG-I and MDA5 during WNV infection

c) RLR signaling during ZIKV infection

B) TLR and MyD88-dependent signaling

16

18

19

20

a) MyD88-dependent signaling restricts WNV replication within the CNS

b) IL-1 signaling promotes WNV clearance from the CNS

20

21

c) TLR signaling through MyD88 plays a minor role in control of WNV infection

22

d) TLR-3 plays a CNS-intrinsic role during WNV infection

e) TLR-3 signaling limits neuroprogenitor growth during ZIKV infection

23

24

C) Type I interferon signaling

a) Type I IFN signaling restricts WNV infection

b) Deficiency in type I IFN signaling as a mouse model for ZIKV infection

25

25

25

D) Type III interferon signaling

a) Type III IFN signaling regulates BBB permeability during WNV infection

b) Type III IFN signaling protects human placental trophoblasts from ZIKV infection

26

27

27

Part 3. Systems biology approaches unravel the host antiviral response

28

A) The antiviral landscape

28

B) Organism level

30

a) Host genetics impacts Influenza A Virus pathogenesis

32

b) Modeling determinants of symptomatic West Nile Virus infection

33

c) Development of an improved small animal pathogenesis model for Ebola Virus infection

34

C) Tissue level

36

a) Transcriptomics uncovers determinants of West Nile virus tissue tropism

36

b) Transcriptomics enhances our understanding of severe Influenza A virus infection

37

c) Transcriptomics defines human antiviral immunity to Dengue Virus

40

D) Cell level

41 a) Single cell transcriptomics 41

b) Single cell RNAseq reveals bimodal expression of immune response genes in dendritic cells

42

c) Single cell analysis reveals subversion of type I IFN production in infected and bystander cells during rotavirus infection

43 d) Outlook for the coming age of single cell analysis 45

E) Conclusion- Systems biology as a tool to chart the antiviral landscape

46

Chapter 2: Systems biology reveals West Nile virus antagonism of STAT5 signaling during infection of human dendritic cells

50

A) Abstract

51

B) Author Summary

52

C) Introduction

53

D) Results

55

E) Discussion

68

F) Experimental procedures

75

G) Figures and legends

82

H) Tables and legends

106

Chapter 3: Zika virus antagonizes type I interferon responses during infection of human dendritic cells

107

A) Abstract

108

B) Author Summary

109

C) Introduction

110

D) Results

113

E) Discussion

130

F) Experimental procedures

138

G) Figures and legends

146

H) Tables and legends

173

Chapter 4: Zika virus infects human placental macrophages

178

A) Abstract

179

B) Introduction

179

C) Results

181

D) Discussion

188

E) Experimental procedures

191

F) Figures and legends

195

G) Tables and legends

207

Chapter 5: General discussion and future directions

211

References

228

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology Biology, Virology
Keyword	Dendritic cells Zika virus Human immune cells Hofbauer cells Flavivirus West Nile virus
Committee Chair / Thesis Advisor	Suthar, Mehul S, Emory University
Committee Members	Grakoui, Arash, Emory University Lowen, Anice Carmen, Emory University Mocarski, Edward S, Emory University Kohlmeier, Jacob, Emory University

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