Transactivation of HIV-1 LTR quasispecies containing specific core/enhancer region binding site polymorphisms by co-linear HIV-1 patient-derived Tat proteins

David Cunningham1,2, Benjamas Aiamkitsumrit1,2, Luna Li1,2, Michael R. Nonnemacher1,2, Vanessa Pirrone1,2, Adam Wojno1,2, Shendra Passic1,2, Brandon Blakey1,2, Jade Ku1,2, Nirzari Parikh1,2, Julio Martin-Garcia1,2, Brian Moldover5, Laila Servance4, David Downie4, Sharon Lewis4,

Jeffrey M. Jacobson1-4, Dennis Kolson6, and Brian Wigdahl1-3

1Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, USA, 2Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, USA, 3Center for Clinical and Translational Medicine, Institute for Molecular Medicine and

Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, USA, 4Division of Infectious Disease and HIV Medicine, Department of Medicine, Drexel University College of Medicine, Philadelphia, PA, USA, 5B-Tech Consulting, Ltd, Philadelphia, PA, USA, 6Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

INTRODUCTION!§  HIV-1 quasispecies contain sequence changes throughout the viral genome. "§  Several HIV-1 genes including tat, env, vpr, and nef, as well as the viral LTR, have

been shown to be involved in the pathogenesis of HIV-related neurological disease. "§  In studies performed with clinical samples derived from HIV-1-infected patients in the

pre-HAART era, single nucleotide changes were observed within the NF-κB-proximal C/EBP and Sp transcription factor binding sites at greater frequency in LTRs isolated from HIV-1-infected patients with more severe disease than those from individuals with less severe HIV disease. "

§  These LTRs contained specific single nucleotide polymorphisms (SNPs) in C/EBP site I (a C-to-T change at position 3 of a consensus B C/EBP binding site I: 3T) that were more frequent in tissues derived from individuals who had died with HAD than those who died without HAD. "

§  It has been reported that specific LTR SNPs within C/EBP site I (3T) and Sp site III (5T) increase in prevalence coordinately over the course of HIV-1 infection and correlate with disease severity."

§  The progression of human immunodeficiency virus type 1 (HIV-1)-associated pathogenesis and disease depends on the ability of the virus to infect and replicate within susceptible cells of the immune and central nervous systems (CNS). "

§  HIV-1-infected monocytes have been shown to be involved in transporting HIV-1 across the blood-brain barrier into the CNS where perivascular macrophages and microglial cells serve as the major populations of cells infected with HIV-1. "

§  Because viral replication in cells of the monocyte-macrophage lineage, including microglial cells, likely plays an important role in the genesis of neurological dysfunction in the absence or presence of antiretroviral therapy, we are interested in defining the cellular and viral factors involved in regulating viral transcription in a number of cellular phenotypes targeted by HIV-1 during the course of infection. "

ABSTRACT!The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) regulates viral gene expression by interacting with multiple viral and host factors, including the viral transactivator protein Tat. We have shown that HIV-1-infected peripheral blood-derived LTR variants containing a 3T CCAAT enhancer binding protein (C/EBP) site I (a C-to-T change at position 3), and/or 5T Sp binding site III (a C-to-T change at position 5) are highly prevalent in late stage disease. LTR sequences derived from longitudinal sampling from patient 107 resulted in the detection of the 3T and 5T co-selected SNPs before the decline of CD4+ T cell counts and decrease in neurological score. Utilizing a 4.4 kb DNA genome fragment, co-linear LTR-driven luciferase reporter clones and full-length Tat expression constructs were derived from longitudinal visits of patient 107 to explore the relative fitness of HIV-1 LTR quasispecies containing these specific sequence alterations and their response to Tat transactivation. Sequence analysis demonstrated that these Tat clones have non-consensus sequence alterations. The results indicated that full-length Tat from patient 107 first return visit (107-R01 Tat101) had higher transactivation capability for its own LTR than the LAI LTR or 107-c1 LTR, suggesting the co-selected Tat was more efficient with its co-linear LTR. Given this, we are currently exploring the co-selected LTR and Tat variations to fully understand if co-selected variations in the LTR and Tat contribute to increased viral transcription and replication resulting in more severe HIV disease."

ACKNOWLEDGEMENTS!This work was funded in part by the Public Health Service, National Institutes of Health through the following grants:"

"

National Institute of Neurological Disorders and Stroke; R01 NS32092"National Institute on Drug Abuse; R01 DA19807"

National Institute of Mental Health; T32 MH079785""

FUTURE DIRECTIONS!

§  Determine if visits R02 and R03 contain alterations in amino acid sequence that effect transactivation ability."

§  Patient 107 has now returned for their R07 visit. Determine the LTR and Tat sequence from the additional longitudinal visits to analyze for genetic variations that correlate to clinical disease progression and LTR and Tat functional alteration."

HIV-1 LTR structure

Figure 1. The LTR regulates HIV-1 viral gene expression via its interaction with multiple viral and host factors. Genetic variation within the binding sites for these factors has been shown to affect viral replication in a cell-type and stimulation-dependent manner. The nucleotide sequence of the region of the ConB U3 that is the focus of the DREXELMED cohort study is indicated.

ATF/

CR

EB

AP-

3

C/E

BP II

C/E

BP I

NF-κB

Sp1

AP-

1

C/E

BP - 405 + 165

TAT TAR P-TEFb

Nuc-0 Nuc-1

TATA

Pol II

TBP

Lef-1

Ets

USF

Oct

-1

Disease severity of patient 107 increases over the four visits

Figure 2. Clinical parameters for patient 107 were collected. Age is presented in years; nadir and current CD4+ T cell count in cells/µl; peak and current viral load in RNA copies/ml; and HAART status as continuous (cH) or discontinuous (dH). The nadir CD4+ T cell count presented in parentheses was the nadir for the patient at all visits until R03 where it dropped to 95 cells/µl. ND = not determined.

Patient Visit Age Yrs

Seropositive Nadir CD4

Current CD4

Peak Viral Load

Current Viral Load

Number of AIDS-defining

illnesses HAART status

Admitted drugs of abuse status

Drugs tested positive for

R00 (visit 1) 43 3 95

(162) 565 455,000 76 0 cH deny any use test negative

R01 (visit 2) 44 4 95

(162) 659 455,000 140 1 cH admit past use test negative

R02 (visit 3) 44 5 95

(162) 720 455,000 64 1 cH admit past use test negative

R03 (visit 4) 45 6 95 95 455,000 154 1 dH admit past use test ND

Patient 107 acquires neurocognitive impairment

A.

B.

Figure 4. (A) The mini bedside neurological exam was modified from the International HIV Dementia Scale. This test screens for short-term memory as well as for concentration and processing speed. (B) Patient 107 was assessed at each visit for neurological impairment. Scores for each visit are presented along with the cube drawn at each visit

Patient-derived HIV-1 LTRs containing the 3T CV/EBP site I and 5T Sp site III exhibit a broad spectrum of basal and Tat-mediated transcription activity in

Jurkat T cells and U-937 monocytic cells

Figure 5. Patient-derived LTR expression constructs (1000 ng) plus and minus HIV-1 IIIB Tat86 expression vector (500 ng) were transiently transfected into 106 (A) Jurkat T cells or (B) U-937 monocytic cells using the FuGene 6 transfection reagent, in parallel with luciferase reporter constructs driven by the HIV-1 LAI LTR and the LAI LTR containing the 5T variant. At 24 h post-transfection, LTR basal and Tat-transactivated transcription activity was detected by the Dual-Luciferase reporter assay; results were presented as the average fold over LAI basal activity in three independent experiments. Patient-derived HIV-1 LTRs containing the 5T Sp site III induced by IIIB Tat presented as the average fold over their own basal activity are presented above each graph. Results shown are derived from transfection performed in triplicate. Mean values (+SD) for each experimental result are shown. Asterisk (*) denotes a statistically significant difference in activity compared to the LAI LTR basal activity with P<0.01.

HIV-1-infected patient 107 Tat transactivates its co-linear patient-

derived LTR better than an unmatched Tat protein in Jurkat T cells

Figure 8. LAI or HIV-1-infected patient 107-derived LTR expression construct (1000 ng) and expression vectors (500 ng) containing IIIB Tat101, HIV-1-infected patient 107-R00-Tat, or 107-R01-Tat were co-transfected into Jurkat T cells. LTR basal and Tat-mediated transcriptional activity was determined 24 h post-transfection by the Dual-Luciferase reporter assay; results were presented as the average fold over LAI basal activity. Representative results shown are derived from transfection experiments performed in triplicate.Each experimental result is presented as a mean value (+SD). An asterisk (*) denotes a statistically significant difference in activity compared to Tat-transactivated LAI LTR with P<0.05. Double asterisks (**) denote a statistically significant difference in activity compared to Tat-transactivated LAI LTR with P<0.01. A space indicates nonconsensus changes among the compared residues.

Patient-derived HIV-1 Tat from patient 107 have nonconsensus variations

compared with IIIB or conB Tat

Figure 9. HIV-1-infected, patient-derived Tat clones from patient 107 were obtained from the 4.4-kb cloned fragments with their co-linear LTRs. Sequence alignments of patient-derived Tat proteins were compared with those from the laboratory strain IIIB and conB Tat. An asterisk (*) indicates an amino acid residue from all visits that align to the conB Tat. A dot (.) indicates conservation of weak groups. A colon (:) indicates conservation of strong groups.

CONCLUSIONS!●  Previous studies in the pre-HAART era have identified the 3T C/EBP site I and 5T

Sp site III to correlate with the severity of HIV disease or be predictive of those HIV-1-infected patients more prone to the development of HIV-1-associated neurological disease

●  Patient 107 showed the 3T and/or 5T SNPs could be identified prior to the onset of disease progression as observed with the decrease in CD4+ T cell count in R03 (compare Figures 2 and 3).

●  In addition, the presence of the 3T and 5T SNPs occurred prior to the decrease in neurocognitive performance in the mini-neurological examination.

●  The results presented demonstrate that different patterns of basal transcription were observed in the two cell lines, suggesting that cellular phenotype may alter the functional properties of 5T- and/or 3T/5T-containing, patient-derived HIV-1 LTRs.

●  In addition, the viral transactivator protein Tat contained genetic variations that demonstrated increased LTR transactivation with its co-linear LTR.

●  Tat genetic variation at position 100 may be predictive of neurocognitive impairment

Patient Visit

C/EBP site II USF Ets Lef-1 ATF/

CREB C/EBP site I

NF-κB site II

NF-κB site I

Sp site III

Sp site II

Sp site I Oct I

A0107-R00 ConB ConB 1A 7A 12A ConB 3T ConB ConB ConB*

(5T) ** ConB ConB 3C

A0107-R01 ConB ConB 1A 7A 12A 6C 3T ConB ConB 5T ConB ConB 3C

A0107-R02 ConB ConB 1A 7A 12A 6C 3T ConB ConB 5T ConB ConB 3C

A0107-R03 ConB 4G ConB 2T 7A ConB 3T 6G ConB ConB ConB ConB ConB ConB

* sequences were evaluated by bioinformatic software ** Sequences were evaluated by 4.4 kb fragment and 454 deep sequencing

vif"

env"vpr"

nef"

LTR"

tat"

rev"

vpu"~4.4 KB"

Figure 6. The 4.4kb HIV-1 fragment amplified and cloned from isolated HIV-1-infected PBMCs to expand sequence analyses from LTR to Env, Tat, and Vpr. To study genetic variation within the HIV-1 genome and how these sequence alterations impact cell- and organ-specific pathogenesis during the course of disease.

Amplification of patient-derived HIV-1 Tat from 4.4kb HIV-1 genome

Figure 7. Two step PCR to amplify full-length HIV-1 Tat. Step 1: PCR the two exons individually using the 4.4kb fragment from clones as template and primers F1 and R1 for exon 1, primers F2 and R2 for exon 2 (R1 and F2 overlap by 17nt). Step 2:, ligate the two PCR products together using the the PCR products from step 1 and primers F1 and R2.

EcoRV

Tat  Exon  1   intron   Tat  Exon  2  

Exon  1   Exon  2  

F1

F1

F2

R1 R2

R2

1st round PCR

2nd round PCR

Kpn I

5’  fragment   3’  fragment  

Figure 3. Sequence variation in each transcription factor binding site from the sequenced LTR PCR product from patient 107 was determined by comparison with the consensus subtype B (conB) LTR determined in Jan of 2002 by the Los Alamos HIV database. The variant is designated by the nucleotide position in that particular binding site and the nucleotide it is changed to. For example if position three of C/EBP site I has a C to T change it is designated as a 3T variant. If there are no changes it is designated as conB.

Single nucleotide polymorphisms in the 12 transcription factor binding sites of

interest for patient 107

Tat AA Position 100 analysis Pt ID R00 R01 R02 R03 R04 R05 R06

37

Time (mnths) between visits 9 10 8 17 6

Neuro NA NA 3.5 ND 10 11 Tat V to H

41

Time (mnths) between visits 9 10 9 5 18 5

Neuro NA NA 4.5 8 5.5 ND ND Tat V to F

51

Time (mnths) between visits 23

Neuro norm 9 Tat conB

56

Time (mnths) between visits 19 8 12 18

Neuro sub 6 9 9 Tat V to C

107

Time (mnths) between visits 14 2 11 6 3 5

Neuro norm 12 8 9.5 10 11.5 10.5 Tat V to K V to K

119

Time (mnths) between visits 36

Neuro norm 5 Tat conB

131

Time (mnths) between visits Neuro 4 Tat V to I

220

Time (mnths) between visits Neuro 11 Tat 3 conB

Figure 10: Sequence analysis of Tat (diagram of protein depicted above) from several patients within the DREXELMED HIV/AIDS Genetic Analysis Cohort revealed variation at position 100 may precede neurocognitive impairment.

101

21 22 37 38 48 49 57 58 72 73

86 87

EXON 1 EXON 2

Transactivation/ CycT1 binding

Co-factor binding CBP/p300

Basic domain/TAR binding domain/ARM

C/EBP binding

Nuclear localization

30

Core Cysteine-rich

domain Acidic domain

Sp1 binding

DNA-PK binding

C C C C C C C LGISYG RKKRRQRRR KKK E E E RGD I II III IV V VI

Basic region

Genetic variation at position 100 of HIV-1 Tat may be predictive of

neurocognitive impairment