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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


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.


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


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


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


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


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.


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:


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


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


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


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.


Are these significant peripheral blood HIV-1 LTR

single nucleotide polymorphisms (SNPs) also

found in HIV-infected brains?

Research Focus #2:


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


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.


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


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


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


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


Frequency of LTR position 168 polymorphism (G to A) with respect to neurocognitive status


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


Frequency of LTR position 251 polymorphism (G to A/C) with respect to neurocognitive status


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


Brain-derived HIV-1 LTR vSNPs at positions 115, 120, 160, and 293 associated with neurocognitive impairment


10%

20%

30%

40%

50%

0% 0%5%

11%

0% 0% 0%

25%

SNP 115A to G/T


10%

20%

30%

40%

50%

8%

0%

11%7%

0% 0%

8%

0%

SNP 120C to T


10%

20%

30%

40%

50%

0% 0%5%

0%0% 0% 0% 0%

SNP 160C to A/G


10%

20%

30%

40%

50%

0% 0%

13%

0%0% 0%

38%

0%

SNP 293G to A/C


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


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


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.


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

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