Table Of ContentPHYLOGENETIC AND PROMOTER ANALYSIS OF THE HUMAN
IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) LONG TERMINAL REPEAT AND
ENVELOPE REGIONS FROM THE TISSUES OF VERTICALLY INFECTED
PATIENTS
By
BRANT ROGER BURKHARDT
JR.
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2001
THIS DISSERTATION IS DEDICATED TO MY WIFE AND PARENTS.
ACKNOWLEDGMENTS
I greatly appreciate all of the training given to me by my mentor, Dr. Maureen
Goodenow. She has been extremely influential in my development as a scientist. I also
thankDr. John Sleasman for providing an important clinical relevance to this project, and
diligent participation in the collection of numerous autopsy specimens that allowed this
study to be possible. 1 greatly appreciate the contributions by Dr. Ayalew Mergia and Dr.
Steven Sugrue for their helpful advice, and constructive criticism throughout this
extensive investigation. I would like to acknowledge all of the members of the
Goodenow lab past and present for theirvaluable help with the completion ofthis project.
In addition to providing me with professional guidance, they have also given me
remarkable friendships and many good times. These members include Deepa Bhatt,
Carter Coberley, James Kohler, Zhong Kou, Feng En Lee, Gina Nykiel, Joseph Oshier,
Steven Pomeroy, Adriana Perez, Brian Peyser, Stephanie Rose, Gregory Taylor, and
Daniel Turtle.
My family and my new wife have been so supportive with my academic
endeavors. I greatly acknowledge my wonderful wife, Patty, for being so caring and
understanding during these stressful times. I also acknowledge my parents, Brant and
Louise Burkhardt, for providing me with so many opportunities to accomplish all ofmy
goals and being such giving parents.
in
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS
iii
LIST OF TABLES vi
LIST OF FIGURES
vii
ABSTRACT
ix
CHAPTERS
INTRODUCTION AND BACKGROUND
1. 1
Specific Aims 1
Significance 5
The Human Immunodeficiency Virus 6
The HTV-1 Long Terminal Repeat (LTR) 7
Phylogentic Analysis ofTissue Specific LTRs 19
Cell-Type Expression Determined by LTR ofotherRetroviruses 20
Cell-Type Specific Expression ofthe HIV-l LTR 20
HIV-1 Envelope 23
Phylogenetic Analysis 25
Specific Aims and Study Design 28
2. PHYLOGENETIC AND PROMOTER ANALYSIS OF THE HIV-1 LTR AND
ENVELOPE REGIONS FROM VARIOUS TISSUES OF VERTICALLY INFECTED
PEDIATRIC PATIENTS 39
Introduction 39
Materials and Methods 41
Results 47
Discussion 55
IV
3. THE THYMUS SERVES AS A SOURCE AND SITE OF EVOLUTION OF T-
CELL LINE TROPIC (CXCR4) AND DUAL-TROPIC (CXCR4±CCR5) ENV
QUASISPECIES 82
Introduction 82
Materials and Methods 85
Results 93
Discussion 100
FUNCTIONAL ANALYSIS OF TISSUE SPECIFIC LTRS USING CONGENIC
4.
RECOMBINANT LUCIFERASE VIRUSES WITHIN PRIMARY CELLS 121
Introduction 121
Materials and Methods 123
Results 133
Discussion 143
CONCLUSIONS
5. 166
REFERENCES
171
BIOGRAPHICAL SKETCH
190
LIST OF TABLES
Table Page
1-1. Overall Patient Cohort andAnalysis Performed 38
2-1. Summary ofClinical Data and Tissues Analyzed from all Patients 62
2-2. V3 Alignment with Frequency, Charge, and Predicted Phenotype 64
3-1. Comprehensive Listing ofAll Patients with Survival Time and Tissues Analyzed by
Env V3 Genotype 104
4-1. MAGI Assay Results from LTR-Luc Recombinant Viruses 152
4-2. Luciferase Experession and Gag Copies from Infection ofPBMC with LTR-Luc
Recombinant X4 Viruses 157
4-3. Luciferase Experession and Gag Copies from Infection ofPBMC with LTR-Luc
Recombinant R5 Viruses 158
4-4. Luciferase Experession and Gag Copies from Infection ofMacrophages with LTR-
Luc Recombinant R5 Viruses 159
4-5. Results ofLTR-Luc Recombinant Virus Infections 165
VI
LIST OF FIGURES
Figure Page
1-1. FflV-1 proviral genome 30
1-2. HIV-1 viral lifecycle as demonstrated by an FttV-1 virion infecting a CD4+ T-
lymphocyte 31
1-3. Generation ofthe HTV-1 LTRs 32
1-4. Major regions ofthe HIV-1 LTR 33
1-5. Transcriptional elements within the HTV-1 LTR U3 region 34
1-6. Functional domains inHTV-1 envelope 35
1-7. Predicted Folding Pattern ofthe HIV-1 gpl20 36
1-8. CellularHost Range ofHIV-1 as Determined by V3 37
2-1. Phylogenetic analysis ofthe DNA sequence from the V3 region ofenvelope and the
LTR from representative tissue sequences from each patient 63
2-2. Nucleotide alignment ofLTR sequences 65
2-3. Parsimony and distance analysis ofthe V3 region ofenvelope 68
2-4. Parsimony and distance analysis ofthe mid-U3 region ofthe LTR 72
2-5. Standard curve ofbulk luciferase 76
2-6. Twenty-four hour time-course ofluciferase expression 77
2-7. Three day time-course experiment ofluciferase expression 78
2-8. Transient transfection ofJurkat cells with patienttissue LTR-luciferase constructs 79
2-9. Transient transfection ofJurkat cells with and without TAT 80
2-10. Transfection ofU937 cells without TAT 81
VII
. 1 1
3-1. Parsimonytree ofthe V3 DNA sequences 105
3-2. Parsimony analysis oftissue V3 sequences 106
3-3. Coreceptor usage and tropism ofenv recombinant viruses 110
3-4. Parsimony analysis ofthe VI amino acid sequences 11
3-5 Parsimony analysis ofthe V2 and C2 amino acid sequences 115
3-6. Phylogenetic analysis ofthe V2-V3 DNA sequences 119
3-7. Model ofT-X4 and D-R5X4 emergence within thymus 120
4-1 Phylogenetic analysis ofthe tissue LTR and V3 regions from Patient C/S 147
4-2. Alignment of LTR sequences 148
4-3. Methodology ofconstruction and measurement ofLTR-Luc Recombinant
Viruses 149
PBMC
4-4. LTR-Luciferase recombinant virus timecourse experiment in 150
4-5. LTR-Luciferase timecourse infection with macrophages 151
4-6. Representative experiment ofPBMC infection with LTR-Luc recombinant viruses. ..153
4-7. PBMC infection with LTR-Luc recombinant viruses from all donors 154
4-8. Representative infection ofmacrophages with LTR-Luc recombinant viruses 155
4-9. Macrophage infection with LTR-Luc recombinant viruses from all donors 156
PBMC
4-10. Luciferase expression per gag copy of and macrophages 160
4-11. Luciferase expression per gag copy ofPBMC infected with LTR-Luc recombinant
X4 andR5 viruses 161
4-12. Relative promoteractivity within different primary cells 162
4-13. Representative experiment ofPBMC infection with Site-Directed LTR-Luc
recombinant viruses 163
4-14. Representative infection ofmacrophages with Site-Directed LTR-Luc recombinant
viruses 164
Vlll
Abstract ofDissertation Presented to the Graduate School
ofthe University ofFlorida in Partial Fulfillment ofthe
Requirements forthe Degree ofDoctor ofPhilosophy
PHYLOGENETIC AND PROMOTER ANALYSIS OF THE HUMAN
IMMUNODEFICIENCY VIRUS TYPE (HIV- LONG TERMINAL REPEAT AND
1 1)
ENVELOPE REGIONS FROM THE TISSUES OF VERTICALLY INFECTED
PATIENTS
By
Brant Roger Burkhardt Jr.
December 2001
Chairman: Maureen M. Goodenow
MajorDepartment: Pathology, Immunology, and Laboratory Medicine
The human immunodeficiency virus type 1 (HIV-1), the etiological agent of
AIDS, displays significant genotypic and phenotypic diversity between and within
patients. This genetic diversity within the virus is dependent on mutation rate ofreverse
transcriptase, host immune response, and other factors produced in the local environment.
HIV-1 infects organs systems that can be either lymphocyte or macrophage dominated;
for example peripheral blood and thymus, or brain and lung, respectively. The primary
determinant for viral entry is envelope (env), whereas the long terminal repeat (LTR)
elements impact gene expression, replication, and integration. HIV has been detected at
high frequency in several tissues; however the genotypic and phenotypic quasispecies
within these tissues have not been evaluated in the context of both env and LTR. The
specific aims of this project were 1) to analyze the env and LTR regions from various
IX
tissues ofvertically infected patients by phylogenetic analysis, 2) to functionally analyze
the tissue specific LTRs by transient transfection in both a T-cell and monocytic cell line,
3) to determine ifthe thymus is the source ofboth T-cell line tropic CXCR4-using (T-
X4), and dual-tropic CXCR4±CCR5-using (D-X4±R5) quasispecies and 4) to assess the
impact of tissue LTRs on gene expression in primary cells such as T-cells and
macrophages using congenic recombinant LTR-Luciferase viruses that are identical
except forthe LTR region.
Env and LTR sequences from brain segregated concordantly from other tissues.
Env and LTR segregation was discordant within the thymus and lung. Tissue specific
segregation oflung sequences was determined by LTR, whereas the env region was the
primary determinant oftissue segregation of thymus sequences. The thymus appears to
be the source ofboth T-X4 and D-X4±R5 quasispecies. Functional analysis by transient
transfection and recombinant LTR congenic viruses demonstrated distinct promoter
differences among tissue specific LTRs within peripheral blood mononuclear cells
(PBMC), macrophages, and a T-cell line. The predominant LTR found in the PBMC had
the highest promoter activity in both macrophages and PBMC. All of the LTRs
examined by recombinant LTR-Luciferase analysis had a much higher promoter
expression in macrophages than PBMC. The lung specific LTR demonstrated a cell type
specific promoter expression in macrophages. Regulatory elements within the LTR can
impact cell-type specific viral gene expression. Tissue and cell-type specific adaptation of
HIV is multifactorial and occurs atthe level ofentry and gene expression that is impacted
by both the env and LTR regions.
