ucalgary_2012_grant_tannika.pdf

UNIVERSITY OF CALGARY
Infection of the Gut by HIV-1: The Pathogenic Role of the Nef Protein
by
Tannika Grant
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
DEPARTMENT OF MEDICAL SCIENCE
CALGARY, ALBERTA
NOVEMBER, 2012
© Tannika Grant 2012

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Abstract
Human Immunodeficiency virus type 1 (HIV-1) targets several cells in the immune
system and induces their progressive decline and the Acquired Immunodeficiency
Syndrome (AIDS). HIV produces viral proteins including Nef; an important
multifunctional protein and viral pathogenic determinant. The gastrointestinal tract and
impairment of its epithelial barrier has been implicated in HIV pathogenesis, thus an
investigation of the contribution of HIV-1 Nef on intestinal barrier dysfunction was
performed. An in vitro model system was established and then used to analyse the
pathogenic role of Nef. Trans-epithelial electrical resistance, Real time (RT)-PCR and
fluorescence microscopy methods were used. It was found that Nef expression in colonic
Caco-2 cells, Jurkat and U937 cells; reduced expression of the Caspases, increased
viability of cells, while decreasing expression of the tight junction proteins. Thus HIV-1
Nef protein may contribute to HIV pathogenesis by disrupting barrier integrity and cell
death of colonic epithelial cells and monolayers.

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Acknowledgements
This work was funded by operating grants from the Canadian Institutes of Health
Research. I would like to thank my supervisor Dr. Guido van Marle for the opportunity to
work in his lab and for the continued support and encouragement in science over the
years. It was a challenge at times, with adversity but what I have learned over the last few
years is immeasurable, and I appreciated your excitement with science and teaching.
I would like to thank my committee members, the department, and Kiran Pandher for
rallying in final months and helping me reach my goal. Kiran I am so thankful for you
going above and beyond to help.
Dr. Karen Massey, I will be forever thankful, for the encouragement and belief, and extra
strength.
Kris Cannon, and Erin Brown thank you so much for the friendship and talks, and
support, in those first two years, you were missed! I would also like to thank Kris Cannon
for the technical support with the pcDNA3 cloning and PCR, and obtaining those last
constructs. I appreciate the support, talks and your enthusiasm and willingness to help.
Frank van der Meer, Rkia Dardari, Mark Chaiyakul, Avril Hatherell, and Sandy
Nishikawa, thank you so much for the talks, frienship and understanding. Cameron, Anna
and Nav, thank you for providing me with fun, friendship, and some great memories.
There have been many others in passing, during my time there and I can’t name all but

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would like to thank those who did help along the way and made positive contributions to
my experience. It can be isolating, lost in the research. I appreciated the moments of
relief, relaxation, of laughter, or just being heard.
To Marinieve Montero and Melita B. Irving, thank you for your knowledge, compassion,
and your strength you have helped in more ways then you know. And I thought of you all
of the time.
Angela Baron, thank you for being a true unconditional friend, dealing with my absences
and inability to come out, but remaining positive, steadfast and understanding. It was
appreciated.
I especially need to thank my parents and sister Traora, for the constant love, support and
encouragement they have provided from the beginning.
I want to thank Braham for the love and supporting me the last few years-even when it
was difficult and we had to live in different cities.
Over all to my family and Braham, for all of the care, love, talks and moving here to
support me during the challenging times. I know that I am so fortunate and I’m in
gratitude and know the true meaning of family.

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Table of Contents
Approval Page ..................................................................................................................... ii
Abstract ............................................................................................................................... ii
Acknowledgements ............................................................................................................ iii
Table of Contents .................................................................................................................v
List of Tables ................................................................................................................... viii
List of Figures and Illustrations ......................................................................................... ix
List of Symbols, Abbreviations and Nomenclature ........................................................... xi
CHAPTER ONE: INTRODUCTION ..................................................................................1
1.1 HIV and AIDS ...........................................................................................................2
1.1.1 HIV-1 Cell Infection ..........................................................................................5
1.2 The HIV-1 Nef Protein ..............................................................................................9
1.2.1 Functions of the Nef Protein ............................................................................10
1.2.2 Effect on Pathogenesis ....................................................................................10
1.3 HIV-1 Pathogenesis and the Gastrointestinal Tract .................................................12
1.3.1 HIV-1 and GI Barrier Dysfunction ..................................................................12
1.3.2 Nef and the GI Tract ........................................................................................14
1.4 The GI Tract .............................................................................................................20
1.4.1 Mucosal Epithelial Layer ................................................................................22
1.4.2 Tight Junctions ................................................................................................23
1.5 The Co Culture Barrier Model .................................................................................24
1.6 Summary ..................................................................................................................26
1.7 Research Hypothesis and Aims ...............................................................................27
CHAPTER TWO: EXPERIMENT METHODS AND MATERIALS ..............................28
2.1 Cell Culture Lines and Bacteria ...............................................................................28
2.1.1 Tissue Culture Cells ........................................................................................28
2.1.2 Bacteria ............................................................................................................30
2.2 Sindbis Virus Expression .........................................................................................31
2.2.1 Production of SINrep5-EGFP ..........................................................................31
2.2.2 Viral Titer and Epithelial Cell Infection ..........................................................32
2.3 pcDNA3 Expression Vector: ...................................................................................33
2.3.1 pcDNA3 Constructs .........................................................................................33
2.3.1.1 PCR Amplification of Nef .....................................................................33
2.3.1.2 Subcloning .............................................................................................35
2.4 Transfection Reactions ............................................................................................37
2.4.1 Lipofection ......................................................................................................37
2.4.2 Neon Electroporation System ..........................................................................38
2.5 Co-Culture System ...................................................................................................41
2.5.1 Indirect Toxicity ..............................................................................................43
2.6 Transepithelial Electrical Resistance Studies ..........................................................44
2.6.1 Direct Toxicity .................................................................................................44
2.6.2 Indirect Toxicity ..............................................................................................44
2.7 Real-time Quantitative PCR (RT-PCR) ...................................................................45
2.7.1 Nucleic Acid Isolation .....................................................................................45