Analysis of metabolic alterations in carbon utilization pathways

113 Pages·2014·6.56 MB·English

by  Krystal Ann Fontaine

Analysis of metabolic alterations in carbon utilization pathways
during virus infection

Krystal Ann Fontaine

A dissertation
submitted in partial fulfillment of the
requirements for the degree of

Doctor of Philosophy

University of Washington
2014

Reading Committee:
Michael Lagunoff, Chair
Adam P. Geballe
Jason G. Smith

Program Authorized to Offer Degree:
Microbiology
©Copyright 2014
Krystal Ann Fontaine
University of Washington

Abstract

Analysis of metabolic alterations in carbon utilization pathways
during virus infection

Krystal Ann Fontaine

Chair of the Supervisory Committee:
Professor Michael Lagunoff
Department of Microbiology

Viruses are dependent on the metabolic machinery of the host cell to supply the energy
and molecular building blocks needed for their replication. Substantial research has focused on
understanding how viruses alter host cellular metabolism in the hopes of identifying metabolic
pathways that are critical for successful infection. In this thesis, we explore how two viruses
important for biodefense, vaccinia virus (VACV) and dengue virus (DENV), manipulate the
global cellular metabolome during infection. In Chapter III, we examine the impact VACV has
on the host metabolic network and discover that VACV implements a strikingly unique carbon
utilization program during infection. Specifically, we define an important role for glutamine
during VACV infection and show that glucose is dispensable for replication. We show that the
glutaminolytic pathway of glutamine metabolism is markedly altered in VACV-infected cells
and is necessary to replenish the TCA cycle during infection. We further demonstrate that
glutaminolysis is required for optimal VACV replication by facilitating the robust viral protein
synthesis needed for infectious virion production. In Chapter IV, we find that DENV
significantly alters glucose metabolism during infection. In particular, we show that DENV
infection activates glycolysis via the upregulation of multiple glycolytic mediators. Moreover,
we demonstrate that the glycolytic pathway is essential for efficient DENV replication.
Therefore, this body of work reveals how viruses dramatically reprogram central carbon
metabolism and offers further evidence that metabolic inhibitors may provide promise in the
treatment of virus infections in the future.
TABLE OF CONTENTS
Page

List of Figures ................................................................................................................................ iii

List of Tables ................................................................................................................................. iv

Chapter I: Introduction .....................................................................................................................1
Viruses and host cellular metabolism ......................................................................................1
Viral-induced changes in central carbon metabolism ..............................................................6
Objectives of this thesis ...........................................................................................................8
Vaccinia virus ..........................................................................................................................9
Dengue virus ..........................................................................................................................12
Hypotheses .............................................................................................................................14

Chapter II: Materials and Methods ................................................................................................17
Cells .......................................................................................................................................17
Inhibitors ................................................................................................................................17
Antibodies ..............................................................................................................................18
Viruses and determination of DENV infection rates .............................................................18
Virus infections for metabolic analysis ..................................................................................19
Cell viability assays ...............................................................................................................20
Nutrient starvation and metabolic inhibitor treatment studies ...............................................20
DENV focus-forming unit reduction assays ..........................................................................22
Glucose uptake assays ............................................................................................................22
Quantitative real-time RT-PCR .............................................................................................23
Western blot analysis .............................................................................................................24
Transmission electron microscopy ........................................................................................25
Statistical analysis ..................................................................................................................26

Chapter III: Vaccinia virus requires glutamine but not glucose
for efficient replication ..................................................................................................................27
Summary ................................................................................................................................27
Introduction ............................................................................................................................28
Results ....................................................................................................................................30
Discussion ..............................................................................................................................36

Chapter IV: Dengue virus requires the induction of glycolysis
for productive infection ..................................................................................................................52
Summary ................................................................................................................................52
Introduction ............................................................................................................................53
Results ....................................................................................................................................55

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Discussion ..............................................................................................................................60

Chapter V: Conclusions and Future Directions .............................................................................74
Summary ................................................................................................................................74
Conclusions ............................................................................................................................75
VACV ................................................................................................................................75
DENV ................................................................................................................................76
Future Directions ...................................................................................................................77
VACV ................................................................................................................................77
DENV ................................................................................................................................79

References ......................................................................................................................................86

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LIST OF FIGURES
Figure Number Page

1-1 Viruses alter carbon metabolism in mammalian cells .......................................................16

3-1 Glutamine metabolism is altered during VACV infection ................................................47

3-2 Glutamine is necessary for optimal infectious VACV production ....................................48

3-3 Glutamine is an essential anaplerotic substrate for the TCA cycle
during VACV infection ......................................................................................................49

3-4 Glutamine is required to maintain the TCA cycle
for VACV protein synthesis ...............................................................................................50

3-5 Glutamine deprivation results in reduced virion formation
in VACV-infected cells ......................................................................................................51

4-1 The glycolytic pathway of glucose metabolism is altered
during DENV infection ......................................................................................................69

4-2 Glutamine metabolism is perturbed in DENV-infected cells ............................................70

4-3 Exogenous glucose is necessary for optimal infectious DENV production ......................71

4-4 Glycolysis is induced during DENV infection ..................................................................72

4-5 Glycolysis is required for maximal DENV replication ......................................................73

5-1 Glutaminolytic enzyme levels are not elevated during VACV infection ..........................84

5-2 HIF-1α mRNA levels are increased during DENV infection ............................................85

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LIST OF TABLES
Table Number Page

3-1 VACV infection alters global host cellular metabolism ....................................................40

4-1 DENV infection alters global host cellular metabolism ...................................................64

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ACKNOWLEDGEMENTS

There are many people I would like to thank for supporting me during my graduate
career. First and foremost, I thank my mentor Michael Lagunoff for his committed guidance in
helping me to become an independent scientist. He provided me with clear direction, yet also
allowed me the freedom to pursue scientific endeavors that I was excited to explore. I would also
like to thank my previous mentors, Sharon Isern and Scott Michael, for the inspiring introduction
to research and for the continuous encouragement they have given me over the years. I would
simply not be where I am today if it were not for these three individuals. I am also indebted to
my committee members (Jason Smith, Adam Geballe, Keith Jerome, and Elaine Raines) for
years of advice and insight. Thank you to all the members of the Lagunoff lab, past and present,
for helping to make my graduate experience such a memorable one. Specifically, I am beyond
thankful for Erica Sanchez and the expansive role she has played in my life. To Roman Camarda,
I will continue to try to find the words to convey just how truly grateful I am that you joined the
lab. I would also like to express my gratitude to my dear friends, including my fellow classmates,
for sharing the path with me on such a monumental journey. Lastly, I am forever grateful for my
family and their ferocious love and endless support. To Daddy, Momma, Gramma, and Pop Pop,
in particular: thank you, thank you, thank you, thank you.

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DEDICATION

I dedicate this dissertation to my Daddy and Momma, the two greatest parents a girl
could have hoped for. How lucky I have been and continue to be to have you always by my side.

To you both, I love you with all of my heart.

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