Discovering Novel Intrinsic Antiviral Responses To Arboviruses

149 Pages·2014·12.4 MB·English

by  Jie Xu

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Publicly Accessible Penn Dissertations
2013
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Jie Xu
University of Pennsylvania, [email protected]
Follow this and additional works at: https://repository.upenn.edu/edissertations
Part of the Allergy and Immunology Commons, Cell Biology Commons, Immunology and Infectious
Disease Commons, Medical Immunology Commons, and the Microbiology Commons
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Xu, Jie, "Discovering Novel Intrinsic Antiviral Responses to Arboviruses: From Transcription to Intestinal
Innate Immunity" (2013). Publicly Accessible Penn Dissertations. 723.
https://repository.upenn.edu/edissertations/723
This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/723
For more information, please contact [email protected].
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Many (re) emerging pathogens are arthropod-borne, transmitted via an insect vector, and cause
significant health and agricultural problems worldwide. Despite their significance, there are few vaccines
and no targeted therapies that exist. This is at least in part due to our limited understanding of virus-host
interactions and the mechanisms used by hosts to restrict infection. In particular, insect vectors play a
critical role in the transmission and spread of these pathogens, but performing molecular and genetic
studies has proven to be difficult. Drosophila is a model organism that shares a high degree of
conservation with insect vectors and has a wealth of molecular and genetic tools for study. Hence, this
thesis aims to provide a deeper understanding of the innate immune factors that restrict arthropod-borne
viruses using this model organism. Using genetic approaches both in vitro and in vivo, two novel antiviral
pathways are discovered and examined in this thesis. First, using RNA interference (RNAi) screening
against disparate viruses in Drosophila, the transcriptional pausing pathway is found to be essential for
antiviral insect immunity. This led to the characterization of a rapidly induced antiviral transcriptional
program, half of which is genetically dependent on this regulatory mechanism and has pausing-
associated chromatin features. These findings suggest transcriptional pausing primes virally induced
genes by enhancing promoter accessibility to allow for rapid gene induction, thereby coordinating a
robust and complex antiviral response. Subsequently, the ERK pathway is found to be part of this
transcriptional response to viral infection. Not only is this nutrient responsive pathway induced by viral
infection, but it also restricts disparate arboviral pathogens. Furthermore, ERK signaling is essential for
antiviral defense in the insect intestinal epithelium. While wild type flies are refractory to oral infection by
arboviruses, this innate restriction can be overcome chemically by oral administration of an ERK pathway
inhibitor or genetically via the specific loss of ERK in the intestinal epithelial cells. In addition, vertebrate
insulin that activates ERK signaling in the mosquito gut during a blood meal, can restrict viral infection in
insect cells and protect against viral invasion of the gut epithelium. These studies collectively
demonstrate that ERK signaling in the insect intestines potently restricts viral infection, suggesting that
insects take advantage of signals in the meal to preemptively activate antiviral immunity.
DDeeggrreeee TTyyppee
Dissertation
DDeeggrreeee NNaammee
Doctor of Philosophy (PhD)
GGrraadduuaattee GGrroouupp
Cell & Molecular Biology
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Sara Cherry
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Frederic Bushman
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Antiviral Immunity, Drosophila melanogaster, Innate Immunity
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Allergy and Immunology | Cell Biology | Immunology and Infectious Disease | Medical Immunology |
Microbiology
This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/723
DISCOVERING
 NOVEL
 INTRINSIC
 ANTIVIRAL
 RESPONSES
 TO
 ARBOVIRUSES:
 FROM

TRANSCRIPTION
 TO
 INTESTINAL
 INNATE
 IMMUNITY

Jie Xu
A DISSERTATION
in
Cell and Molecular Biology
Presented to the Faculties of the University of Pennsylvania
in
Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
2013

Supervisor of Dissertation
________________________
Dr. Sara Cherry
Associate Professor of Microbiology

Graduate Group Chairperson
________________________
Dr. Daniel S. Kessler, Associate Professor of Cell and Developmental Biology
Dissertation Committee
Dr. Nancy Bonini, Professor of Biology
Dr. Margaret Chou, Associate Professor of Pathology and Laboratory Medicine
Dr. Gary Koretzky, Francis C. Wood Professor of Medicine
Dr. Frederic Bushman, Professor of Microbiology (Chair)
“DISCOVERING NOVEL INTRINSIC ANTIVIRAL RESPONSES TO ARBOVIRUSES: FROM
TRANSCRIPTION TO INTESTINAL INNATE IMMUNITY”
COPYRIGHT
2013
Jie Xu

This work is licensed under the
Creative Commons Attribution-
NonCommercial-ShareAlike 3.0
License

To view a copy of this license, visit
http://creativecommons.org/licenses/by-ny-sa/2.0/
ii
DEDICATION
I would like to dedicate this dissertation to my grandparents: Xu Zhong Liang, Yang Jian
Qun, Li Jian, and Gao Ke Qin. Each of one their lives is a complex story of hardship and
perseverance. Most importantly, it is from each of their lives that inspire my own
academic and life aspirations.

iii
ACKNOWLEDGMENTS
I would like to thank the following individuals for their contributions to this dissertation:

Dr. Sara Cherry for providing unequivocal mentorship throughout my graduate training,
which has singlehandedly helped shape me as a scientific researcher in almost every
aspect, as well as for her valuable guidance and support in my personal life.

My thesis committee members Dr. Gary Koretzky, Dr. Nancy Bonini, Dr. Frederick
Bushman, and Dr. Margaret Chou for their mentorship and guidance throughout my
training.

Dr. Gregory Grant for his kindness and forming a wonderful collaboration for the crucial
bioinformatics analyses in Chapter III.

Dr. Matt Tudor for performing all of the transcriptional profiling analyses discussed in
Chapter III and the concluding remarks, along with critical reading of manuscripts.

Dr. Gerd Blobel for helpful advice and support in Chapter III, as well as Rena Zheng for
helping with the technical aspects of chromatin immunoprecipitation (ChIP) performed in
Chapter III.

Dr. Beth Gordesky-Gold for her contributions in fly husbandry and experiments for both
Chapters III and IV. The undergraduates, particularly Veronica Schad and Kendrick
Chow, for also helping to maintain the fly room and always willing to help others.

Dr. Michele Markstein for critical reading of the manuscript in Chapter IV as well as
providing important technical advice for manipulating fly guts for microscopy.

Dr. Leah Sabin for performing the RNAi screen and validation of the initial factors
identified for Chapters III and IV.

Kaycie Hopkins and Ian Lamborn for helping screen the three MAPK pathways against
disparate viruses for antiviral activity, as discussed in Chapter IV.

Ari Yasunaga for providing crucial technical support in all of the phospho-ERK westerns
in Chapter IV, the insulin experiments in cell culture and flies, as well as the receptor
experiments in the concluding remarks.

Gregory Osborn for helping screen the 65 MAPK-dependent genes for antiviral activity
against disparate viruses, as discussed in the concluding remarks.

All members of the Cherry lab, including Ryan Moy, Sheri Hanna, Debasis Panda, for
helpful ideas and making the lab such a special place to do research.

My family and friends for their encouragement, guidance, and love.

The Howard Hughes Medical Institute (HHMI) for providing financial support in my
graduate training, as well as providing opportunities to further explore my research
interests.
iv
ABSTRACT

DISCOVERING NOVEL INTRINSIC ANTIVIRAL RESPONSES TO ARBOVIRUSES:
FROM TRANSCRIPTION TO INTESTINAL INNATE IMMUNITY
Jie Xu
Sara Cherry
Many (re) emerging pathogens are arthropod-borne, transmitted via an insect vector,
and cause significant health and agricultural problems worldwide. Despite their
significance, there are few vaccines and no targeted therapies that exist. This is at least
in part due to our limited understanding of virus-host interactions and the mechanisms
used by hosts to restrict infection. In particular, insect vectors play a critical role in the
transmission and spread of these pathogens, but performing molecular and genetic
studies has proven to be difficult. Drosophila is a model organism that shares a high
degree of conservation with insect vectors and has a wealth of molecular and genetic
tools for study. Hence, this thesis aims to provide a deeper understanding of the innate
immune factors that restrict arthropod-borne viruses using this model organism. Using
genetic approaches both in vitro and in vivo, two novel antiviral pathways are discovered
and examined in this thesis. First, using RNA interference (RNAi) screening against
disparate viruses in Drosophila, the transcriptional pausing pathway is found to be
essential for antiviral insect immunity. This led to the characterization of a rapidly
induced antiviral transcriptional program, half of which is genetically dependent on this
regulatory mechanism and has pausing-associated chromatin features. These findings
suggest transcriptional pausing primes virally induced genes by enhancing promoter
accessibility to allow for rapid gene induction, thereby coordinating a robust and complex
antiviral response. Subsequently, the ERK pathway is found to be part of this
transcriptional response to viral infection. Not only is this nutrient responsive pathway
induced by viral infection, but it also restricts disparate arboviral pathogens.
Furthermore, ERK signaling is essential for antiviral defense in the insect intestinal
epithelium. While wild type flies are refractory to oral infection by arboviruses, this innate
restriction can be overcome chemically by oral administration of an ERK pathway
inhibitor or genetically via the specific loss of ERK in the intestinal epithelial cells. In
v
addition, vertebrate insulin that activates ERK signaling in the mosquito gut during a
blood meal, can restrict viral infection in insect cells and protect against viral invasion of
the gut epithelium. These studies collectively demonstrate that ERK signaling in the
insect intestines potently restricts viral infection, suggesting that insects take advantage
of signals in the meal to preemptively activate antiviral immunity.

vi
TABLE OF CONTENTS
CHAPTER 1 Introduction
1. (Re) emerging Infectious Arboviral Pathogens ...................................................... 1
2. The Infection Cycle in Insect Vectors ..................................................................... 3
3. Antiviral Vector Immunity: Tissue Barriers and Immune Compartments ................ 4
4. Cellular Antiviral Immune Mechanisms in Insect Vectors ...................................... 7
5. Drosophila melanogaster as a Model Organism for Antiviral Innate Immunity ...... 14
6. Aims of the Present Studies ................................................................................... 18

CHAPTER 2 Materials and Methods
1. Cells and Cell Culture ............................................................................................ 27
2. Viruses ................................................................................................................... 27
3. Antibodies .............................................................................................................. 27
4. dsRNA Synthesis ................................................................................................... 28
5. RNA interference .................................................................................................... 28
6. Viral Infections for Cell Culture Experiments .......................................................... 28
7. Immunofluorescence .............................................................................................. 29
8. Immunoblotting ....................................................................................................... 29
9. Adult Fly Infections ................................................................................................. 30
10. RNA, Northern Blotting and RT-qPCR ................................................................... 31
11. Chromatin-Immunoprecipitation (ChIP) ................................................................. 32
12. DNA Microarray Analysis ...................................................................................... 32
13. Bioinformatics and Statistics .................................................................................. 33
14. Oligonucleotide Sequences for RT-qPCR .............................................................. 34

CHAPTER 3. Transcriptional Pausing Orchestrates a Rapid Antiviral Response in
Drosophila melanogaster
1. Abstract .................................................................................................................. 36
2. Background ............................................................................................................ 36
3. Results ................................................................................................................... 38
4. Discussion .............................................................................................................. 46
5. Figures ................................................................................................................... 49
6. Supplemental Figures ............................................................................................ 56
7. Supplemental Tables ............................................................................................ 62

CHAPTER 4. Erk Signaling Couples Nutrient Status with Antiviral Defense in the Insect
Gut
1. Abstract .................................................................................................................. 88
2. Background ............................................................................................................ 88
3. Results ................................................................................................................... 89
4. Discussion .............................................................................................................. 93
5. Figures ................................................................................................................... 95
6. Supplemental Tables ............................................................................................ 99
7. Supplemental Figures ............................................................................................ 100

CHAPTER 5. Concluding Remarks. ............................................................................ 113

CHAPTER 6. References ............................................................................................ 122

vii