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Viral Promoter Polymorphisms in HIV Disease
Gregory C. Antell2013 Sigma Xi Research Showcase
March 15, 2013
Graduate StudentDrexel University
School of Biomedical Engineering, Science, and Health Systems
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Multiple factors influence HIV-1 pathogenesis and HIV-1-associated neurocognitive disorders (HAND)
HIV-associated neurocognitive disorders include HIV-associated
dementia (HAD) and minor cognitive motor disorder (MCMD).
A spectrum of cellular targets are vulnerable to infection, which may lead to physiological compartmentalization and
tissue-specific selective pressures.
Pathogenesis is shaped by the host immune
response and genetics, drug therapy, drug abuse, and aging.
HAND
Host & Therapy
Viral
Cellular
Molecular diversity emerges in the virus as it adapts to selective
pressures. Particular variants may serve as biomarkers of advanced
neurological disease.
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
HIV-1 pathogenesis and associated neurological dysfunction
CNS viral evolution
CNS viral entryExtra-CNS viral evolution
Brain
Blood
BloodBrainBarrier
Acute Infection Clinical Latency AIDS / Dementia
CD4 count > 500 200-500 < 200
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Brain
resident
microglia
cells
neurons
HIV-1-infected
perivascular
macrophage
HIV-1 CNS entry and infection of resident cell populations
astrocytes
Viral gene products have neurotoxic
effects on astrocytes and
neurons
Mucosal compartment
Bone marrow compartment
Peripheral Blood
Lymphoid compartment
Other end organsBlood-BrainBarrier
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
HIV-1 replication scheme
genomic RNA
mRNA
Viral GeneExpression
Assembly
integratedproviral DNA
Nuclear Transportand Integration
Protein synthesisand processing
Budding
Reverse Transcription
gp120
CD4
CoreceptorsCCR5CXCR4
Absorption and entry
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
The HIV-1 genome and 5’-LTR organization
5’LTR 3’LTRgagpol env
vifvpr
vpu nef
revtat
U3455 552 638
nt1
HS2 HS3 HS4nuc-1nuc-0 +1-245-405 +20 +165
leaderR U5
Core regionModulatory regionEnhancer region
NFAT
ATF/
CREB
C/EB
P US
1NF
-kB
Sp1
TBP
LBP-
1
AP-1
AP3-
LIR
F
Sp1
AP-1
AP-4
LEF-
1
Ets-
1
Usf
GR
NFAT
c-M
yb
AP-1
AP-1
ATF/
CREB
LSF
C/EB
P US
2
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Background and demographics of the DREXELMED HIV/AIDS Genetic Analysis Cohort
Visit Patients Seen
Initial Visit 503
First Return 298
Second Return 202
Third Return 136
Fourth Return 95
Fifth Return 67
Sixth Return 43
Seventh Return 29
Eighth Return 17
Ninth Return 7
Tenth Return 2
Total 1399
Demographic Variables Categories Count (%)/Mean (+/- SD)
with clinical variables
GenderMale 332 (66%)Female 169 (33.6%)
Race
Black/AA 418 (83.1%)White 62 (12.3%)Other (AI/AN, multiple, asian) 16 (3.2%)
Unknown 7 (1.4%)
HAART statuscH 424 (84.3%)dH 43 (8.5%)nH 34 (6.7%)
Age 45.43 (± 8.569)Years since diagnosed 11.916 (± 7.312)
Patients enrolled in the DrexelMed Cohort are recruited from the Philadelphia region and are scheduled to return every six months. At each visit, a patient interview is conducted, a blood sample is collected, and a neurological exam is performed.
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
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?
Research Focus #1:
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Whole BloodIsolation of genomic DNA
PCR product sequencing
Ficoll-Pacque Plus gradient
Serum & PBMC separation
Sequenceanalysis
BSL-3 Facility
HIV-1 Sequence Database
Qiagen DNEasy Tissue Kit
Gel extraction
PCR amplify proviral DNASeparate on agarose gel
Incubate with Taq to add A
overhang
pCR4-TOPO
PCR amplify/ clone proviral DNA
pGL3 Basic
Functional analysis
Serum and cell bankingClinical and virus/host genomic data management
Luminex Human Cytokine 30 plex
Serum
Viromic analysis of DREXELMED HIV/AIDS Genetic Analysis Cohort in the HAART era
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
HIV-1 LTR SNP densities in patients from the DREXELMED HIV/AIDS Genetic Analysis Cohort
1 15 29 43 57 71 85 99 113
127
141
155
169
183
197
211
225
239
253
267
281
295
309
323
337
351
365
379
393
407
421
435
449
463
477
491
505
519
533
547
561
575
589
603
617
631
0
100
200
300
400
500
600
700
800
900
0
100
200
300
400
500
600
700
Sequence Coverage SNP Density
Nucleotide Position on ConB (Jan 2002) Reference Sequence
Cov
erag
e (N
umbe
r of S
eque
nces
)
SN
P D
ensi
ty (N
umbe
r of m
utat
ions
)
• 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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Significant LTR SNPs
Phenotype Position Ref./Mut. Mutant Freq Effect p-value
CD4 Count
108 A/CGT 38.0% -41.228 0.0176120 C/AT 6.2% 72.950 0.0200181 A/CG 8.3% -72.320 0.0173293 G/ACT 11.4% -46.920 0.0452
Viral Load
108 A/CGT 38.0% 184.4% 0.0010115 A/GT 18.5% 60.7% 0.0301160 C/AG 6.3% 46.7% 0.0278168 G/ACT 14.8% 60.2% 0.0282251 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.
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Are these significant peripheral blood HIV-1 LTR
single nucleotide polymorphisms (SNPs) also
found in HIV-infected brains?
Research Focus #2:
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Autopsy tissue punches QIAGEN DNeasy Tissue Procedure
Isolation of genomic DNA
PCR amplify proviral
DNA
Separate on agarose gel
Preparation for sequencing and
sequence analysis
Nested PCR amplifies LTR from proviral DNA
HIV-1 Brain LTR Sequence Database
Isolation of HIV-1 brain-derived LTRs for sequence analysis
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Number of brain and spleen sequences used in analysis
Brain Normal Subsyndromic MCMD HADSequence Number 16 18 95 38Patient Number 2 2 14 6Spleen
Normal Subsyndromic MCMD HADSequence Number 3 2 19 7Patient Number 2 2 14 4
Patient samples availableHAD 6MCMD 16Subsyndromic 3Normal 2Uninfected 1TOTAL 28Tissue regions availableCerebellum 28Deep White Matter 28Head of Caudate 28Midfrontal Gyrus 28Parietal 28Thalamus 27Spleen 23
National NeuroAIDS Tissue Consortium – University of TexasDirector: Ben Gelman, M.D., Ph.D.
Autopsy tissue samples were collected from multiple brain sites, as well as spleen, from patients with
varying degrees of neurological impairment.
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Prevalence of clinically significant peripheral blood LTR SNPs in HIV-1 infected brain tissue
Nucleotide Position
TF Site
Number of individuals
Total in Spleen
Total in Brain
Neuro.Normal
Neuro.Impaired
108 COUP/ AP1 20 11 75 13 73
115 COUP/ AP1 5 1 6 0 7
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Clinically significant peripheral blood HIV-1 LTR SNPs are found in all regions of the HIV-1-infected brain except for SNP 181
108 115 120 160 168 181 251 2930
2
4
6
8
10
12
14
16
CerebellumDeep White MatterHead of CaudateMidfrontal GyrusParietalThalamus
LTR SNP Position
Num
ber o
f SN
Ps
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
HIV-1 LTR SNPs identified in the peripheral blood are also found to associate with neurologic impairment in the brain
Nucleotide Position
Found in Brain? TF Site Texas Cohort Notes DREXELMED PBMC
Notes
108 Yes COUP/AP1 Decreases with impairment
Decreased CD4 countIncreased viral load
115 Yes COUP/AP1 Only in impairment Increased viral load
120 Yes COUP Mostly in impairment Increased CD4 count
160 Yes AP1 Rare, only found in brain and impairment Increased viral load
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Frequency of LTR position 108 polymorphism (A to G) with respect to neurocognitive status
NORMAL SUBSYNDROMIC MCMD HAD0%
10%20%30%40%50%60%70%80%90%
100% 92% 90%
63%54%
100%
50%46%
50%BRAIN SPLEEN
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Frequency of LTR position 168 polymorphism (G to A) with respect to neurocognitive status
NORMAL SUBSYNDROMIC MCMD HAD0%
5%
10%
15%
20%
25%
0% 0%
8%
18%
0% 0%
8%
0%
BRAINSPLEEN
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Frequency of LTR position 251 polymorphism (G to A/C) with respect to neurocognitive status
NORMAL SUBSYNDROMIC MCMD HAD0%
5%
10%
15%
20%
25%
0% 0%
16%14%
0% 0%
23%
0%
BRAINSPLEEN
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Brain-derived HIV-1 LTR vSNPs at positions 115, 120, 160, and 293 associated with neurocognitive impairment
NORMAL SUBSYNDROMIC MCMD HAD0%
10%
20%
30%
40%
50%
0% 0%5%
11%
0% 0% 0%
25%
SNP 115A to G/T
NORMAL SUBSYNDROMIC MCMD HAD0%
10%
20%
30%
40%
50%
8%
0%
11%7%
0% 0%
8%
0%
SNP 120C to T
NORMAL SUBSYNDROMIC MCMD HAD0%
10%
20%
30%
40%
50%
0% 0%5%
0%0% 0% 0% 0%
SNP 160C to A/G
NORMAL SUBSYNDROMIC MCMD HAD0%
10%
20%
30%
40%
50%
0% 0%
13%
0%0% 0%
38%
0%
SNP 293G to A/C
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
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
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
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.
I N S T I T U T E F O R M O L E C U L A R M E D I C I N E A N D I N F E C T I O U S D I S E A S E
Brian Wigdahl, Ph.D., Professor & Chair
Department of Microbiology & ImmunologyDrexel University College of Medicine
William Dampier, Ph.D.Rui Feng, Ph.D.Jeffrey Jacobson, M.D.Pooja Jain, Ph.D.Steve Jennings, Ph.D.Zafar Khan, Ph.D.Sandhya Kortagere, Ph.D.Fred Krebs, Ph.D. Michele Kutzler, Ph.D.David Libon, M.D.Julio Martin-Garcia, Ph.D.Brian Moldover, Ph.D.
Olimpia Meucci, M.D., Ph.D.Sonia Navas-Martin, Ph.D.Michael Nonnemacher, Ph.D.Vanessa Pirrone, Ph.D.Laura Steel, Ph.D.Nirzari Parikh, M.S.Shendra Passic, M.S.Benjamas AiamkitsumritGreg AntellBrandon BlakeyJessica BrownNatalie Chen
Director
Betty CondranJessica CrossSatinder DahiyaDavid DownieBrian FrantzArchana GuptaNneka IkpezeShawn KeoganChristina KolliasSharon LewisRaphael LukovAndrea Partridge
NINDS NIDA
Renzo PeralesMatt RimbeyGermaine RivalFiorella RossiSonia ShahLuz Jeanette SierraMarianne StrazzaGokul SwaminathanKen ThompsonCristian ValenciaJean WilliamsWen Zhong
NIAIDNCINIMH