Functional and Structural Mimicry of A-Kinase Anchoring Proteins by Human Adenovirus E1A

220 Pages·2017·6.05 MB·English

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Cason R. King, The University of Western Ontario
Supervisor: Mymryk, Joe S, The University of Western Ontario
A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree
in Microbiology and Immunology
© Cason R. King 2018
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King, Cason R., "Functional and Structural Mimicry of A-Kinase Anchoring Proteins by Human Adenovirus
E1A" (2018). Electronic Thesis and Dissertation Repository. 5286.
https://ir.lib.uwo.ca/etd/5286
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Abstract
As an obligate intracellular parasite, human adenovirus (HAdV) must utilize host factors
for survival and replication. Early during infection, its multifunctional E1A protein
interacts with an impressive range of cellular target proteins to exert control over the
cellular environment. Through these virus-host interactions, E1A massively reprograms
both viral and cellular transcription to activate the other HAdV genes, downregulate the
host’s immune response, and induce the cell cycle. Consequently, E1A converts the
infected cell into a compliant state more amenable for HAdV replication, resulting from
its numerous protein-protein interactions. I sought to examine E1A’s interaction with
cellular protein kinase A (PKA), a well-studied component of host cAMP signaling that
plays a critical role in cellular transcription. While characterizing the molecular
determinants of the E1A-PKA interaction, I showed that E1A uses mimicry of cellular A-
kinase anchoring proteins (AKAPs) to usurp PKA for the benefit of HAdV. This
mechanism allowed E1A to outcompete endogenous AKAPs and induce an E1A-
mediated relocalization of PKA into the nucleus of infected cells. Here, PKA was
recruited to viral gene promoters. This ultimately resulted in increased viral transcription
from these loci as well as enhanced protein synthesis and higher levels of viral
replication. This mechanism of mimicry was conserved in E1A proteins across various,
distinct species of HAdV, indicating its evolutionary importance. Interestingly, different
E1A proteins displayed a preference for manipulating one type of PKA isoform over
another. Despite mechanistic differences, all species of HAdV that commandeered PKA
via their E1A proteins required this action for wild-type levels of both viral genome
replication and infectious progeny production. Additionally, these E1A proteins could
induce transcription from a cAMP- and PKA-regulated reporter gene suggesting that this
interaction may modulate host genes in a manner similar to viral ones. Together, these
studies show that manipulation of PKA by E1A is a conserved feature of diverse HAdVs.
This allows these viruses to subvert cellular cAMP signaling and use a host factor to
enhance multiple aspects of their replication cycles. Furthermore, this mechanistically
demonstrates HAdV E1A as the first known viral AKAP – a unique form of viral
mimicry.
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Keywords

Human adenovirus, HAdV, Early region 1A, E1A, Protein kinase A, PKA, A-kinase
anchoring protein, AKAP, Cyclic AMP, cAMP, Viral mimicry, Viral AKAP, Protein-
protein interaction, Gene expression, Subcellular localization, Transcription, Viral
replication, Oncoprotein, Hub protein
Co-Authorship Statement
Sections of Chapter 1 of this thesis were published in Trends in Microbiology, 2016;
24:323–4 or submitted to mBio (mBio00390-18). I was the primary author of both these
manuscripts.
Chapter 2 of this thesis was published in PLoS Pathogens, 2016; 12:e1005621. I
performed all the experiments with the exception of the molecular modelling in Figures
2.3A–G and 2.4 which was carried out by Brennan Dirk in the laboratory of Dr. Jimmy
Dikeakos.
Chapter 3 of this thesis was published in Journal of Virology, 2018; 92(8):e01902-17. I
was the primary author and performed all the experiments.
Chapter 4 of this thesis contains unpublished data. The RNAseq experiment and initial
analysis was carried out by Drs. Mike Cohen and Greg Fonseca. I performed all
subsequent data analyses described in this section.
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Acknowledgments
I am faced with the daunting task of succinctly thanking a massive number of terrific
people without whom none of this would have been possible. Rest assured the names that
appear below deserve far more gratitude from me than a few pages could convey.
First and foremost I am immensely thankful for my boss, mentor, and friend, Dr. Joe
Mymryk. Years ago he sparked my interest in virology research by being a charismatic
and approachable undergraduate lecturer. From there, my journey through science has
been guided by his incredible knowledge, brilliant ideas, and wonderful personality. I am
truly lucky to have spent my formative scientific years in his lab learning not only the ins-
and-outs of molecular biology, but also the finer points of what makes a truly good
person. The selfless way Joe finds happiness in helping his students succeed is a lesson I
can never forget.
I would like to thank my other professional mentors, Drs. Lakshman Gunaratnam and
Greg Dekaban for years of valuable advice and criticism of my work, culminating in this
thesis. I am also grateful to other members of the Department of Microbiology &
Immunology, particularly Drs. Jimmy Dikeakos and Kelly Summers who evolved from
being superiors and examiners into collaborators and friends.
At the bench, I had an abundance of riches when it came to senior students willing to
teach me. Drs. Matt Miller, Greg Fonseca, and Mike Cohen stand out both as teachers of
key techniques and examples of personal success I’ve tried to emulate. London family
and friends (both in and out of the department) are too numerous to list, but I feel
compelled to mention how their longtime presence in my professional and personal life
motivated me even when experiments failed. From my cousin Danny to my friends
Tanner, Ali, Steve, Jeremy, Kyle, Rod, Lee, Jordan, Sam, Joe, Alan, Stu, and especially
Kris James: thanks for everything.
My family is an unrelenting source of encouragement and I am blessed to have an
amazing support network. My parents, Sherry and Doug, mean more to me than the
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deepest, most heartfelt words could explain. Thank you for always enabling my
endeavours and helping me make good decisions. Lastly, the man who taught me to keep
my stick on the ice both in hockey and in life; thank you for showing all of us what true
strength and character are. While I miss you terribly and wish you were here, I know I
have made you proud…
This one’s for you, Grandpa.
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Table of Contents
Abstract ................................................................................................................................ i
Co-Authorship Statement .................................................................................................... ii
Acknowledgments .............................................................................................................. iii
Table of Contents ................................................................................................................ v
List of Tables ...................................................................................................................... x
List of Figures .................................................................................................................... xi
List of Appendices ........................................................................................................... xiv
List of Abbreviations ........................................................................................................ xv
Chapter 1 ............................................................................................................................. 1
1 Introduction .................................................................................................................... 1
1.1 General Introduction ............................................................................................... 1
1.2 Adenovirus .............................................................................................................. 3
1.2.1 Physical properties of adenoviruses ............................................................ 3
1.2.2 HAdV life cycle .......................................................................................... 6
1.2.3 HAdV transcripts ........................................................................................ 7
1.2.4 HAdV diversity and virulence .................................................................. 10
1.3 HAdV Early Region 1A (E1A) ............................................................................. 14
1.3.1 E1A gene, transcripts, and protein products ............................................. 14
1.3.2 E1A is a viral hub protein ......................................................................... 16
1.3.3 E1A can act as either an oncoprotein or tumour suppressor ..................... 18
1.3.4 E1A as a tool for identifying eukaryotic regulatory proteins.................... 20
1.3.5 E1A modulates viral transcription ............................................................ 23
1.3.6 E1A modulates cellular transcription ........................................................ 27
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1.4 Protein Kinase A (PKA) ....................................................................................... 29
1.4.1 Discovery and structure of PKA ............................................................... 29
1.4.2 Cellular functions of PKA......................................................................... 32
1.4.3 A-kinase anchoring proteins (AKAPs) ..................................................... 34
1.5 Viral Mimicry ....................................................................................................... 36
1.6 Thesis Overview ................................................................................................... 37
1.6.1 Chapter 2: Functional and structural mimicry of cellular protein kinase
A anchoring proteins by a viral oncoprotein ............................................. 37
1.6.2 Chapter 3: Mimicry of cellular A kinase anchoring proteins is a
conserved and critical function of E1A across various human
adenovirus species .................................................................................... 38
1.6.3 Chapter 4: General Discussion .................................................................. 38
1.7 References ............................................................................................................. 39
Chapter 2 ........................................................................................................................... 70
2 Functional and structural mimicry of cellular protein kinase A anchoring proteins
by a viral oncoprotein ................................................................................................... 70
2.1 Introduction ........................................................................................................... 70
2.2 Materials and Methods .......................................................................................... 72
2.2.1 Cell lines and transfections ....................................................................... 72
2.2.2 Virus infection of cells .............................................................................. 72
2.2.3 RNAi knockdown ..................................................................................... 72
2.2.4 Plasmids .................................................................................................... 74
2.2.5 Western blotting and co-immunoprecipitation ......................................... 74
2.2.6 Immunofluorescence microscopy and image analysis .............................. 76
2.2.7 Quantitative RT-PCR ................................................................................ 76
2.2.8 Chromatin immunoprecipitation ............................................................... 76
2.2.9 Statistical analysis ..................................................................................... 80
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2.2.10 Docking methods ...................................................................................... 80
2.3 Results ................................................................................................................... 81
2.3.1 Multiple PKA subunits are conserved targets of HAdV E1A .................. 81
2.3.2 The sequence of E1A that binds PKA resembles a cellular AKAP .......... 84
2.3.3 E1A binds the same surface of PKA targeted by cellular AKAPs ........... 86
2.3.4 E1A functions as a viral AKAP ................................................................ 90
2.3.5 E1A alters PKA subcellular localization .................................................. 94
2.3.6 E1A uses PKA to enhance HAdV gene expression ................................ 101
2.3.7 E1A uses PKA to enhance viral replication ............................................ 106
2.4 Discussion ........................................................................................................... 108
2.5 References ........................................................................................................... 115
Chapter 3 ......................................................................................................................... 121
3 Mimicry of cellular A-kinase anchoring proteins is a conserved and critical
function of E1A across various human adenovirus species ....................................... 121
3.1 Introduction ......................................................................................................... 121
3.2 Materials and Methods ........................................................................................ 123
3.2.1 Cell lines and transfections ..................................................................... 123
3.2.2 Viruses and infection of cells .................................................................. 123
3.2.3 RNAi knockdown ................................................................................... 124
3.2.4 Plasmids .................................................................................................. 124
3.2.5 Western blotting and co-immunoprecipitation ....................................... 127
3.2.6 Immunofluorescence microscopy ........................................................... 127
3.2.7 Virus replication assay ............................................................................ 127
3.2.8 Luciferase reporter assay ........................................................................ 131
3.2.9 PKA activity assay .................................................................................. 131
3.2.10 Statistical analysis ................................................................................... 131
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3.3 Results ................................................................................................................. 132
3.3.1 Protein kinase A subunits are conserved binding targets of the N-
terminal region of E1A across numerous HAdV species ....................... 132
3.3.2 E1A proteins that bind PKA use an AKAP-like sequence motif ............ 134
3.3.3 E1A binds to PKA via the PKA docking-dimerization domain ............. 134
3.3.4 Distinct E1A proteins relocalize PKA regulatory subunits in a type-
specific manner ....................................................................................... 138
3.3.5 E1A’s ability to act as a vAKAP is modular and malleable by
mutagenesis ............................................................................................. 146
3.3.6 Interaction with PKA is required for full E1A-mediated transactivation
of a PKA-driven promoter ...................................................................... 150
3.3.7 HAdV infection stimulates the cAMP pathway ...................................... 153
3.3.8 Requirement of PKA for viral replication is conserved across
numerous HAdV species ......................................................................... 156
3.4 Discussion ........................................................................................................... 159
3.5 References ........................................................................................................... 165
Chapter 4 ......................................................................................................................... 170
4 General Discussion ..................................................................................................... 170
4.1 Thesis Summary .................................................................................................. 170
4.2 The interaction between E1A and PKA .............................................................. 171
4.3 Mimicry of cellular AKAPs by E1A .................................................................. 172
4.4 Viral retasking of PKA to enhance infection ...................................................... 173
4.5 Conservation of viral AKAPs ............................................................................. 175
4.6 Usurping PKA to alter host cell transcription ..................................................... 177
4.7 Contribution of E1A-PKA interaction to oncogenic transformation .................. 180
4.8 Prevalence of cellular scaffold mimics in the viral universe .............................. 181
4.9 Concluding remarks ............................................................................................ 182
viii
4.10 References ........................................................................................................... 183
Appendices ...................................................................................................................... 191
Curriculum Vitae ............................................................................................................ 195
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