CHARACTERIZING EPITHELIAL RESPONSES TO ROTAVIRUS INFECTION IN THE GUT Open Access

Frias, Amena H. (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/ht24wj516?locale=pt-BR%2A
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Abstract


CHARACTERIZATION OF EPITHELIAL RESPONSES TO
ROTAVIRUS INFECTION IN THE GUT
By Amena H. Frias

Rotavirus (RV) is the leading cause of diarrhea in young children worldwide. RV
targets intestinal epithelial cells (IEC), which clear infection within 7 days via pathways
not strictly dependent on adaptive immunity. We hypothesized that IEC sense RV via
innate pattern-recognition receptors (PRRs) and mount anti-viral immune responses
involving Type 1 IFN-α/β. We sought to determine if RV structural components induce
epithelial anti-viral gene expression and define the role of type 1 IFN in the RV-induced
innate immune response. Model epithelia (HT29 IEC) were treated with RV (MOI 1-10),
UV-irradiated RV (UV-RV) which was non-infectious but structurally intact, RV
components including purified dsRNA and nucleic-acid free capsid shells (also called
virus-like particles, or VLPs), and RV in presence of neutralizing antibodies to IFN-α/β.
Gene transcription was assessed using microarray or qRT-PCR. Viral protein and anti-
viral marker expression (STAT1, IRF 3/7, PKR), as well as PARP cleavage and DNA
fragmentation (apoptotic events), were detected via western blotting or immunostaining.
Secretion of IFN-β and IL-8 was measured using ELISA, and cell morphology was
observed under a light microscope. Epithelia stimulated apically with trypsinized
(protease-treated) RV exhibited upregulation of anti-viral markers including IFN-β but
not IFN-α, and these trends were largely mimicked by UV-RV. RV dsRNA and VLPs
poorly induced anti-viral signaling in comparison to UV-RV. Blockade of IFN-β
signaling dramatically abrogated RV-induced anti-viral gene expression and prevented
apoptosis. Surprisingly, impaired IFN-β activity also correlated with modest suppression
of viral protein synthesis, particularly in a PKR-dependent manner. These data suggest
IEC detect RV components via a trypsin-dependent, apical pathway of infection, and
subsequently activate Type 1 IFN responses that promote anti-viral signaling, apoptosis,
and viral replication in infected cells.

Table of Contents

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION 1

1.1 Intestinal epithelial response to bacteria 2

1.2 Epidemiology of rotaviruses 3

1.3 Rotavirus structure and replication 4

1.3.1 Rotavirus structure 4

1.3.2 Rotavirus replication 5

1.4 Rotavirus pathogenesis 7

1.4.1 Rotaviral induction of diarrhea 7

1.4.2 Extraintestinal spread of rotavirus 8

1.4.3 Genetic reassortment of rotavirus 9

1.5 Rotavirus vaccine development 10

1.5.1 Live, attenuated rotavirus vaccines 10

1.5.2 Inactivated rotavirus and VLP vaccines 11

1.6 Adaptive and innate immune response to rotavirus 12

1.6.1 Experimental animal models 12

1.6.2 Role of type 1 IFNs in innate anti-rotaviral immunity 14

1.6.3 Intestinal epithelial response to rotavirus 15

1.7 Cellular anti-viral defense mechanisms 15


CHAPTER 2: INTESTINAL EPITHELIA ACTIVATE ANTI-VIRAL
SIGNALING VIA INTRACELLULAR SENSING OF ROTAVIRUS

COMPONENTS 18

Abstract 19

Introduction 20

Results 24

Discussion 30

Materials and Methods 34

Tables 40

Figures 43

Figure Legends 53

CHAPTER 3: ROTAVIRUS-INDUCED IFNS PROMOTE ANTIVIRAL
SIGNALING, APOPTOSIS AND REPLICATION IN INTESTINAL
EPITHELIAL CELLS 58

Abstract 59

Introduction 60

Results 63

Discussion 68

Materials and Methods 72

Tables 78

Figures 81

Figure Legends 86

CHAPTER 4: DISCUSSION 89

REFERENCES 98

LIST OF TABLES
(listed in order of appearance as they appear in the chapter)


CHAPTER 2

2-1 Fold change induction of anti-viral markers relative to mock 40

2-2 Genes induced by UV-RV, RV VLPs, and RV RNA
relative to mock 41

2-3 Fold change induction of anti-viral markers relative to mock 42

CHAPTER 3

3-1 Percent Threshold of Fluorescence in RV-infected cells vs.
RV-infected cells with anti-IFN a/b 78

3-2 Viral levels in supernatants of RV-infected cells vs. RV-infected

cells with anti-interferon antibodies 79

3-3 Percent Threshold of Fluorescence in RV-infected cells vs.
RV-infected cells treated with 2AP 80

LIST OF FIGURES

(listed in order of appearance as they appear in the chapter)

CHAPTER 2

2-1 Anti-viral protein expression in RV-infected intestinal epithelia 43

2-2 Anti-viral protein expression exhibited in RV-infected and UV-RV
stimulated epithelia 44

2-3 Anti-viral protein expression exhibited in epithelia treated with RV
and UV-RV in the presence or absence of trypsin 45

2-4 Anti-viral protein expression exhibited in epithelia treated apically and

basolaterally with UV-RV 46

2-5 Transcription profiles of epithelia stimulated with RV, UV-RV, and RV

in the presence of Type 1 IFN (a/b) antibodies 47

2-6 Anti-viral protein expression exhibited in epithelia treated with UV-RV,

RV VLPs and RV RNA 48

2-7 RV, UV-RV, and VLP cell entry during early stages of infection 49

S2-1 Supplementary Figure 50

CHAPTER 3

3-1 RV-induced type I IFN, particularly IFN-b, elicits epithelial anti-viral

gene expression 81

3-2 RV-induced type I IFN induces apoptosis in intestinal epithelia 83

3-3 RV-induced type I IFNs promote, not impair, viral protein synthesis
and cell-to-cell spread 84

3-4 Rotaviral spread is PKR-dependent 85

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