Nucleotide Modifications of RNA Suppress RIG-I Antiviral Signaling by Unique Mechanisms The ...

141 Pages·2016·3.19 MB·English

Nucleotide Modifications of RNA Suppress RIG-I
Antiviral Signaling by Unique Mechanisms
Citation
Durbin, Ann M. 2016. Nucleotide Modifications of RNA Suppress RIG-I Antiviral Signaling by
Unique Mechanisms. Doctoral dissertation, Harvard University, Graduate School of Arts &
Sciences.
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Nucleotide modifications of RNA suppress RIG-I antiviral signaling by unique mechanisms

A dissertation presented
by
Ann Fiegen Durbin
to
The Division of Medical Sciences
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
in the subject of
Virology

Harvard University
Cambridge, Massachusetts

April 2016
© 2016 Ann Fiegen Durbin
All rights reserved.
Dissertation Advisor: Professor Lee Gehrke Ann Fiegen Durbin

Nucleotide modifications of RNA suppress RIG-I antiviral signaling by unique mechanisms
Abstract
In order to counter pathogen infection while preventing autoimmune responses, the human
innate immune system must be precisely regulated to distinguish “self” from “non-self”. Pattern
recognition receptors detect “non-self” pathogen RNAs and initiate antiviral signaling.
Accumulated evidence suggests that host “self” RNAs contain modified nucleotides that evade
or suppress immune signaling; however, the precise mechanisms are not understood. Defining
these mechanisms is relevant toward understanding the biology of immunity as well as the
applied use of RNAs as therapeutic molecules, where reducing ligand immunogenicity is
essential. Evidence from our lab and others’ suggests that the cytosolic RNA helicase RIG-I
(retinoic acid inducible gene-I) detects not only the 5’ terminus and double-stranded nature of
RNA, but also the presence/absence of modified nucleotides. In the present study, we use a
model RNA ligand (polyU/UC), derived from the 3’ untranslated region of the hepatitis C virus
RNA, to dissect the mechanisms by which RNAs containing nucleotide modifications suppress
or evade RIG-I signaling. Five assays were developed to test our hypothesis that eight different
nucleotide modifications, both natural and synthetic, share a common mechanism of innate
immune evasion. In vitro transcribed 5’-triphosphate polyU/UC RNA containing canonical
nucleotides potently activates the RIG-I signaling pathway in transfected cells, culminating in an
antiviral state. When transcribed with any of eight modified nucleotides, the polyU/UC RNA
suppressed the RIG-I antiviral response. Unexpectedly, the modified nucleotides had different
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effects on RIG-I:RNA binding affinity, as well as RIG-I conformational change. The data
suggest that multiple RIG-I evasion/suppression mechanisms associated with different modified
nucleotides may have evolved to effect a common result of interrupting innate immune signaling
responses to “self” RNA. Our findings hold implications for understanding the co-evolution of
the innate immune response and RNA modification pathways across domains of life, as well as
for defining approaches for testing the multitude of naturally occurring and synthetic nucleotides
that may have utility in the design of therapeutic RNAs.

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Contents
Abstract .......................................................................................................................................... iii
Acknowledgements ....................................................................................................................... vii
Chapter 1: Introduction ................................................................................................................... 1
Pattern Recognition Receptors of the Innate Immune System .................................................... 2
Innate Immune System and Auto-immunity ............................................................................... 6
Modified RNA Detection by the Innate Immune System ........................................................... 9
Biology of Post-Transcriptional RNA Modifications ............................................................... 10
Applications in RNA Therapeutics ........................................................................................... 13
Research Objective .................................................................................................................... 14
References ................................................................................................................................. 16
Chapter 2: RNA Transcription and Purification ........................................................................... 24
Introduction ............................................................................................................................... 25
Materials and Methods .............................................................................................................. 27
Results ....................................................................................................................................... 32
Discussion ................................................................................................................................. 42
References ................................................................................................................................. 43
Chapter 3: Cell-based Assays of RIG-I Signaling ........................................................................ 46
Introduction ............................................................................................................................... 47
Materials and Methods .............................................................................................................. 49
Results ....................................................................................................................................... 54
Discussion ................................................................................................................................. 61
References ................................................................................................................................. 62
Chapter 4: Biochemical Assay for RIG-I Interaction with RNA .................................................. 64
Introduction ............................................................................................................................... 65
Materials and Methods .............................................................................................................. 68
Results ....................................................................................................................................... 73
Discussion ................................................................................................................................. 83
References ................................................................................................................................. 85
Chapter 5: Discussion ................................................................................................................... 88
Preface ....................................................................................................................................... 89
Limitations of This Work .......................................................................................................... 90
Mechanistic Classification of RNA Modifications ................................................................... 94
Implications for Evolution of Innate Immune Detection of RNA ............................................ 97
v
Applications for RNA Therapeutics ........................................................................................ 100
Closing Remarks ..................................................................................................................... 103
References ............................................................................................................................... 104
Appendix: Dengue Virus Infection of Primary Human Hepatocytes ......................................... 109
Preface ..................................................................................................................................... 110
Introduction ............................................................................................................................. 110
Materials and Methods ............................................................................................................ 114
Results ..................................................................................................................................... 117
Discussion ............................................................................................................................... 126
References ............................................................................................................................... 128

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Acknowledgements
I am deeply grateful to the many individuals who have supported me during my graduate
work: The members of my Dissertation Advisory Committee, Jon Kagan, Michaela Gack, and
Karl Münger, for their constructive criticism and enthusiasm as my project has developed; my
labmates, especially John Lian and Irene Bosch, who have provided comradery across the years.
I am grateful to the many bright scientists with whom I have collaborated, who have each
patiently shared their expertise and time: Sangeeta Bhatia, Robert Schwartz, Kathleen Christine,
Charlotte Odendall, Chen Wang, and Joe Marcotrigiano, and Anton McCaffrey. I am thankful to
Trilink Biotechnologies for providing a set nucleoside triphosphates for the project.
I am grateful for the support of the students and faculty of the Virology program, and to
my close friends in BBS and Immunology programs. I am grateful to the students past and
present of Science in the News, for striving to bring science to a wider audience. SITN provided
me with opportunities to improve my communication skills, and share my enthusiasm with a
community of scientists and non-scientists.
I am grateful to my family, especially my parents and my sisters, who have supported my
personal and scientific aspirations with words of encouragement, always reminding me to ‘be
powerful’ during the most difficult times.
I am especially grateful to my husband, Wil, for his many sacrifices, to join me in
Boston, to enable my long days and weekends in lab, to nourish me with rich meals cooked with
love, to serenade me with his sincere songs, to share with me an awe of the natural world, to
comfort me in my times of disaster and doubt, to believe in me and my scientific work without
hesitation, and to build a meaningful, thoughtful, and loving life together.
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I am forever bound in gratitude to my advisor and mentor, Lee Gehrke, who has always
led by example: in designing and conducting experiments with integrity, treating colleagues with
genuine respect and magnanimity, and balancing scientific pursuits with a warm and full life at
home. He was my steadfast guide and undaunted supporter in the most difficult times: when the
technical obstacles seemed insurmountable, when the data seemed unintelligible, when my
energy and optimism were drained. He was my resolute advocate and enthusiastic enabler in the
high times: pioneering new methods, discovering enticing observations, synthesizing novel
conclusions, and presenting my science to the broader community. I humbly aspire to embody in
my own life, in the next chapter beyond the lab, the admirable character of my mentor Lee.

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Chapter 1: Introduction