1. Viral Promoter Polymorphisms
in HIV Disease
Gregory C. Antell
2013 Sigma Xi Research Showcase
March 15, 2013
Graduate Student
Drexel University
School of Biomedical Engineering, Science, and Health Systems

2. The HIV epidemic has neurological consequences
 An average of 6,800 new HIV infections and 5,700 HIV-related
deaths occur daily worldwide
 Infection of the central nervous system occurs in approximately
80% of infected individuals
 Approximately 50% of HIV-infected adults and children will
demonstrate a neurological disorder at one time
 The advent of anti-retroviral therapy has diminished the incidence
of HIV-associated neurocognitive disorders to a lesser extent than
other AIDS-related diseases
 Prevalence of neurocognitive diseases has actually increased
due to the prolonged survival of HIV infected individuals
 Neuropathology of HIV disease remains largely unknown and a
critical area of current and future research
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3. Multiple factors influence HIV-1 pathogenesis and HIV-1-
associated neurocognitive disorders (HAND)
Host & Therapy Cellular
Pathogenesis is shaped A spectrum of cellular targets are
by the host immune vulnerable to infection, which may lead to
response and genetics, physiological compartmentalization and
drug therapy, drug tissue-specific selective pressures.
abuse, and aging.
HAND
Viral
Molecular diversity emerges in the
virus as it adapts to selective HIV-associated neurocognitive
pressures. Particular variants may disorders include HIV-associated
serve as biomarkers of advanced dementia (HAD) and minor
neurological disease. cognitive motor disorder (MCMD).
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4. HIV-1 pathogenesis and associated neurological dysfunction
CNS viral evolution Brain
Blood
Brain
Barrier
CNS viral entry
Extra-CNS viral evolution Blood
 HIV-1 likely enters the brain during acute infection and during the absence
of effective therapy or immune dysfunction
 In the brain resident microglial cells and perivascular macrophages are the
major cellular targets for infection
 Release of viral and cellular neurotoxic mediators results in the alteration of
the blood-brain barrier and neuronal dysfunction
Acute Infection Clinical Latency AIDS / Dementia
CD4 count > 500 200-500 < 200
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5. HIV-1 CNS entry and infection of resident cell populations
Peripheral Blood Brain
Mucosal compartment HIV-1-infected
perivascular
macrophage
Lymphoid compartment
resident
microglia
cells
Bone marrow compartment Viral gene products have
neurotoxic effects on
astrocytes and neurons
Blood-
Other end organs Brain
Barrier astrocytes neurons
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6. HIV-1 replication scheme
 HIV-1 replication is controlled by the viral promoter, termed the long terminal repeat (LTR), as
well as the regulatory genes Tat and Vpr
 While HIV-1 is known to have an entry phenotype, it is hypothesized that it may also have
distinct replication phenotypes that associates with particular host cell phenotypes and/or
physiological compartments
Absorption and entry
gp120
CD4 Budding
Coreceptors
CCR5
CXCR4 genomic Assembly
Reverse RNA
Transcription
Protein synthesis
Nuclear Transport Viral Gene and processing
and Integration Expression
integrated
proviral DNA mRNA
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7. The HIV-1 genome and 5’-LTR organization
tat
vpr
rev
5’LTR gag vif 3’LTR
pol vpu env nef
455 552 638
nt1 leader
U3 R U5
nuc-0 HS2 HS3
+1
nuc-1 HS4
-405 -245 +20 +165
Modulatory region Core region
Enhancer region
 The HIV-1 genome is flanked by two LTR sequences: the 5’-LTR and the 3’-LTR
 The 5’-LTR acts as the promoter for viral gene expression
 The LTR contains a high concentration of binding sites for cellular transcription factors, which
can vary according to the host cell phenotype
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8. Background and demographics of the DREXELMED HIV/AIDS
Genetic Analysis Cohort
Patients enrolled in the DrexelMed Cohort are recruited from the Patients
Philadelphia region and are scheduled to return every six months. Visit
Seen
At each visit, a patient interview is conducted, a blood sample is
collected, and a neurological exam is performed. Initial Visit 503
First Return 298
Demographic Count (%)/Mean (+/- SD) Second Return 202
Categories
Variables with clinical variables
Male 332 (66%) Third Return 136
Gender
Female 169 (33.6%) Fourth Return 95
Black/AA 418 (83.1%)
Fifth Return 67
White 62 (12.3%)
Race Other (AI/AN, multiple, Sixth Return 43
16 (3.2%)
asian)
Seventh Return 29
Unknown 7 (1.4%)
cH 424 (84.3%) Eighth Return 17
HAART status dH 43 (8.5%) Ninth Return 7
nH 34 (6.7%)
Tenth Return 2
Age 45.43 (± 8.569)
Years since diagnosed 11.916 (± 7.312) Total 1399
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9. Research Focus #1:
Do specific HIV-1 LTR single nucleotide
polymorphisms (SNPs) derived patient
peripheral blood samples correlate with
alterations in clinical HIV disease
parameters in the HAART era?
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10. Viromic analysis of DREXELMED HIV/AIDS Genetic Analysis
Cohort in the HAART era
Ficoll-Pacque Qiagen DNEasy
Whole Blood Plus gradient Tissue Kit
Serum & PBMC
separation Luminex Human PCR amplify
BSL-3 Facility Cytokine 30 plex proviral DNA
Separate on
Serum and cell banking agarose gel
Clinical and virus/host genomic data management
HIV-1 Sequence Database
Sequence PCR product
analysis sequencing
Gel extraction
pGL3 Basic pCR4-TOPO
Incubate with
PCR amplify/ Taq to add A
Functional overhang
analysis clone proviral DNA
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11. HIV-1 LTR SNP densities in patients from the DREXELMED
HIV/AIDS Genetic Analysis Cohort
900 700
Sequence Coverage
800
SNP Density (Number of mutations)
SNP Density 600
Coverage (Number of Sequences)
700
500
600
500 400
400 300
300
200
200
100
100
0 0
115
381
400
134
153
172
191
210
229
248
267
286
305
324
343
362
419
438
457
476
495
514
533
552
571
590
609
628
20
39
58
77
96
1
Nucleotide Position on ConB (Jan 2002) Reference Sequence
• LTR SNP coverage and frequency was calculated for 461 patients and 1127
sequences
• SNPs are observed throughout the LTR sequence and can be mapped to
transcription factor binding sites
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12. Nine HIV-1 LTR SNPs associate with change in CD4 count and log
viral load away from the average of the cohort
 The single nucleotide polymorphisms (SNPs) identified from patient peripheral
blood samples can be plotted according to base pair position in the LTR and
association with CD4+ T cell count and log viral load
 Data is adjusted for patient age, sex, and race
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13. Significant LTR SNPs
Phenotype Position Ref./Mut. Mutant Freq Effect p-value
108 A/CGT 38.0% -41.228 0.0176
120 C/AT 6.2% 72.950 0.0200
CD4 Count
181 A/CG 8.3% -72.320 0.0173
293 G/ACT 11.4% -46.920 0.0452
108 A/CGT 38.0% 184.4% 0.0010
115 A/GT 18.5% 60.7% 0.0301
Viral Load 160 C/AG 6.3% 46.7% 0.0278
168 G/ACT 14.8% 60.2% 0.0282
251 G/ACT 8.8% 53.9% 0.0315
A total of 9 SNPs, located at 8 distinct nucleotide positions, were identified to associate
with the clinical parameters of CD4+ T cell count and/or viral load at a statistically
significant level (p-vale < 0.05). The effect in this case is defined as the change in
CD4+ T cell count or the percent change in viral load away from the average.
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14. Research Focus #2:
Are these significant peripheral blood
HIV-1 LTR single nucleotide
polymorphisms (SNPs) also found in
HIV-infected brains?
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15. Isolation of HIV-1 brain-derived LTRs for sequence analysis
Nested PCR amplifies LTR
Autopsy tissue punches QIAGEN DNeasy
from proviral DNA
Tissue Procedure
PCR
Isolation of amplify
genomic DNA proviral
DNA
HIV-1 Brain LTR Sequence Database Separate on agarose gel
Preparation for
sequencing and
sequence analysis
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16. Number of brain and spleen sequences used in analysis
Patient samples available Brain
HAD 6 Normal Subsyndromic MCMD HAD
MCMD 16 Sequence
Subsyndromic 3 Number 16 18 95 38
Normal 2 Patient
Uninfected 1 Number 2 2 14 6
TOTAL 28 Spleen
Tissue regions available Normal Subsyndromic MCMD HAD
Cerebellum 28 Sequence
Deep White Matter 28 Number 3 2 19 7
Head of Caudate 28 Patient
Midfrontal Gyrus 28 Number 2 2 14 4
Parietal 28
Thalamus 27 Autopsy tissue samples were collected from multiple
Spleen 23 brain sites, as well as spleen, from patients with
varying degrees of neurological impairment.
National NeuroAIDS Tissue Consortium – University of Texas
Director: Ben Gelman, M.D., Ph.D.
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17. Prevalence of clinically significant peripheral blood LTR SNPs
in HIV-1 infected brain tissue
Nucleotide TF Number of Total in Total in Neuro. Neuro.
Position Site individuals Spleen Brain Normal Impaired
COUP/
108 20 11 75 13 73
AP1
COUP/
115 5 1 6 0 7
AP1
120 COUP 5 1 10 1 10
160 AP1 2 0 3 0 3
168 unk 8 1 10 0 11
181 unk 4 4 0 0 4
251 unk 10 4 13 0 17
293 USF 8 5 8 0 13
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18. Clinically significant peripheral blood HIV-1 LTR SNPs are found in
all regions of the HIV-1-infected brain except for SNP 181
16
14
12
Number of SNPs
Cerebellum
10
Deep White Matter
8 Head of Caudate
Midfrontal Gyrus
6
Parietal
4 Thalamus
2
0
108 115 120 160 168 181 251 293
LTR SNP Position
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19. HIV-1 LTR SNPs identified in the peripheral blood are also
found to associate with neurologic impairment in the brain
Nucleotide Found in
TF Site Texas Cohort Notes DREXELMED PBMC Notes
Position Brain?
Decreases with Decreased CD4 count
108 Yes COUP/AP1
impairment Increased viral load
115 Yes COUP/AP1 Only in impairment Increased viral load
120 Yes COUP Mostly in impairment Increased CD4 count
Rare, only found in
160 Yes AP1 Increased viral load
brain and impairment
168 Yes unk Only in impairment Increased viral load
181 No unk Only found in spleen Decreased CD4 count
251 Yes unk Only in impairment Increased viral load
293 Yes USF Only in impairment Decreased CD4 count
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20. Frequency of LTR position 108 polymorphism (A to G) with
respect to neurocognitive status
100% 92% 100% 90%
90%
80%
70% 63%
60% 54%
50% 50% BRAIN
50% 46%
40% SPLEEN
30%
20%
10%
0%
NORMAL SUBSYNDROMIC MCMD HAD
BRAIN SPLEEN
Nucleotide Normal Subsyndromic MCMD HAD Nucleotide Normal Subsyndromic MCMD HAD
A (reference) 1 1 24 13 A (reference) 0 1 7 2
G (mutation) 11 9 40 15 G (mutation) 2 1 6 2
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21. Frequency of LTR position 168 polymorphism (G to A) with
respect to neurocognitive status
25%
20% 18%
15%
10% 8% 8%
5%
0% 0% 0% 0% 0% BRAIN
0%
SPLEEN
BRAIN SPLEEN
Normal Subsyndromic MCMD HAD Normal Subsyndromic MCMD HAD
G (reference) 12 10 54 24 G (reference) 2 2 10 4
A (mutation) 0 0 10 4 A (mutation) 0 0 3 0
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22. Frequency of LTR position 251 polymorphism (G to A/C) with
respect to neurocognitive status
25% 23%
20%
16%
15% 14%
BRAIN
10%
SPLEEN
5%
0% 0% 0% 0% 0%
0%
NORMAL SUBSYNDROMIC MCMD HAD
BRAIN SPLEEN
Normal Subsyndromic MCMD HAD Normal Subsyndromic MCMD HAD
G (reference) 12 10 54 24 G (reference) 2 2 10 4
A/C (mutation) 0 0 10 4 A/C (mutation) 0 0 3 0
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23. Brain-derived HIV-1 LTR vSNPs at positions 115, 120, 160,
and 293 associated with neurocognitive impairment
50% 50%
SNP 115 SNP 120
40% 40%
A to G/T C to T
30% 30%
25%
20% 20%
11% 11%
8% 8% 7%
10% 5%
10%
0% 0% 0% 0% 0% 0% 0% 0% 0%
0% 0%
NORMAL SUBSYNDROMIC MCMD HAD NORMAL SUBSYNDROMIC MCMD HAD
50% 50%
SNP 160 SNP 293 38%
40% 40%
C to A/G G to A/C
30% 30%
20% 20%
13%
10% 5% 10%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
0% 0%
NORMAL SUBSYNDROMIC MCMD HAD NORMAL SUBSYNDROMIC MCMD HAD
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24. Summary of major findings
• Eight HIV-1 LTR SNPs derived from peripheral blood
mononuclear cells associate with change in CD4 count and/or
log viral load away from the average of the cohort
• Clinically significant peripheral blood HIV-1 LTR SNPs are found
in all regions of the HIV-1-infected brain except for SNP 181
• HIV-1 LTR SNPs identified in the peripheral blood are also
found to associate with neurologic impairment in the brain,
particularly SNPs 108, 168, and 251
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25. Future directions
• Identify SNPs in PBMC-derived LTR sequence that correlate
with neurological disease and determine if they are present in
HIV-1-infected brain tissue
• Identify SNPs in brain-derived LTR sequences that associate
with neurological impairment, and assess their presence in
PBMC-derived LTRs
• Analyze additional HIV genes that contribute to proviral
transcription, such as Tat and Vpr, for single nucleotide
polymorphisms that correlate with clinical parameters
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26. Ultimate objective of this research
To identify a panel of genetic variants in the
proviral HIV-1 LTR (or other parts of the genome)
derived from PBMCs that are predictive of
neurologic decline
We envision a scenario where a simple blood test and diagnostic
PCR can cue physicians about potential problems and treatment
strategies. This viral SNP marker panel would be used in tandem
with other neurocognitive biomarkers.
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27. Brian Wigdahl, Ph.D., Professor & Chair
Department of Microbiology & Immunology
Drexel University College of Medicine
Director
William Dampier, Ph.D. Olimpia Meucci, M.D., Ph.D. Betty Condran Renzo Perales
Rui Feng, Ph.D. Sonia Navas-Martin, Ph.D. Jessica Cross Matt Rimbey
Jeffrey Jacobson, M.D. Michael Nonnemacher, Ph.D. Satinder Dahiya Germaine Rival
Pooja Jain, Ph.D. Vanessa Pirrone, Ph.D. David Downie Fiorella Rossi
Steve Jennings, Ph.D. Laura Steel, Ph.D. Brian Frantz Sonia Shah
Zafar Khan, Ph.D. Nirzari Parikh, M.S. Archana Gupta Luz Jeanette Sierra
Sandhya Kortagere, Ph.D. Shendra Passic, M.S. Nneka Ikpeze Marianne Strazza
Fred Krebs, Ph.D. Benjamas Aiamkitsumrit Shawn Keogan Gokul Swaminathan
Michele Kutzler, Ph.D. Greg Antell Christina Kollias Ken Thompson
David Libon, M.D. Brandon Blakey Sharon Lewis Cristian Valencia
Julio Martin-Garcia, Ph.D. Jessica Brown Raphael Lukov Jean Williams
Brian Moldover, Ph.D. Natalie Chen Andrea Partridge Wen Zhong
NINDS NIMH NCI NIDA NIAID
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