This document summarizes a study that analyzed genetic variations in the HIV-1 genome from a patient over time. Key findings include: 1. Specific single nucleotide polymorphisms (SNPs) in transcription factor binding sites of the HIV-1 LTR, including a C-to-T change at position 3 of the C/EBP site (3T) and a C-to-T change at position 5 of the Sp site (5T), were detected in samples from patient 107 before declines in CD4 count and neurological score. 2. A Tat protein derived from an early sample from patient 107 (107-R01 Tat) more strongly transactivated the patient's own LTR compared to a

1. 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
ABSTRACT ! Single nucleotide polymorphisms in the Amplification of patient-derived HIV-1 Genetic variation at position 100 of
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
12 transcription factor binding sites of Tat from 4.4kb HIV-1 genome HIV-1 Tat may be predictive of
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
interest for patient 107 tat
"
neurocognitive impairment
rev
"
C-to-T change at position 3), and/or 5T Sp binding site III (a C-to-T change at position Patient C/EBP ATF/ C/EBP NF-κB NF-κB Sp Sp Sp
USF Ets Lef-1 Oct I vif
" nef
"
Visit site II CREB site I site II site I site III site II site I
5) are highly prevalent in late stage disease. LTR sequences derived from longitudinal vpr
" env
" LTR"
A0107- ConB* Basic domain/
sampling from patient 107 resulted in the detection of the 3T and 5T co-selected SNPs ConB ConB 1A 7A 12A ConB 3T ConB ConB ConB ConB 3C Cysteine-rich
R00 (5T) ** Acidic domain TAR binding
vpu"
domain Core domain/ARM EXON 1 EXON 2
before the decline of CD4+ T cell counts and decrease in neurological score. Utilizing a A0107-
ConB ConB 1A 7A 12A 6C 3T ConB ConB 5T ConB ConB 3C
~4.4 KB"
4.4 kb DNA genome fragment, co-linear LTR-driven luciferase reporter clones and full- R01 21 22 30 37 38 48 49 57 58 72 73 101
A0107- Figure 6. The 4.4kb HIV-1 fragment amplified and cloned from isolated HIV-1-infected C
C
C
C
C
C
C
LGISYG
RKKRRQRRR
RGD
KKK
E E
E
length Tat expression constructs were derived from longitudinal visits of patient 107 to R02
ConB ConB 1A 7A 12A 6C 3T ConB ConB 5T ConB ConB 3C
PBMCs to expand sequence analyses from LTR to Env, Tat, and Vpr. To study genetic I
II
III
IV
V
VI
explore the relative fitness of HIV-1 LTR quasispecies containing these specific A0107- variation within the HIV-1 genome and how these sequence alterations impact cell- and
ConB 4G ConB 2T 7A ConB 3T 6G ConB ConB ConB ConB ConB ConB Basic region 86 87
sequence alterations and their response to Tat transactivation. Sequence analysis R03
organ-specific pathogenesis during the course of disease. Transactivation/
CycT1 binding Nuclear
demonstrated that these Tat clones have non-consensus sequence alterations. The * sequences were evaluated by bioinformatic software localization
Co-factor binding C/EBP
results indicated that full-length Tat from patient 107 first return visit (107-R01 Tat101) ** Sequences were evaluated by 4.4 kb fragment and 454 deep sequencing Kpn I
F1
F2
CBP/p300 binding DNA-PK
had higher transactivation capability for its own LTR than the LAI LTR or 107-c1 LTR, Figure 3. Sequence variation in each transcription factor binding site from the Sp1
binding
suggesting the co-selected Tat was more efficient with its co-linear LTR. Given this, we 1st round PCR
Tat
Exon
1
intron
Tat
Exon
2
binding
sequenced LTR PCR product from patient 107 was determined by comparison with the
are currently exploring the co-selected LTR and Tat variations to fully understand if co- consensus subtype B (conB) LTR determined in Jan of 2002 by the Los Alamos HIV R1
R2
EcoRV
selected variations in the LTR and Tat contribute to increased viral transcription and database. The variant is designated by the nucleotide position in that particular binding
Tat AA Position 100 analysis
replication resulting in more severe HIV disease." site and the nucleotide it is changed to. For example if position three of C/EBP site I 5’
fragment
3’
fragment
Pt ID R00 R01 R02 R03 R04 R05 R06
Time (mnths)
9 10 8 17 6
has a C to T change it is designated as a 3T variant. If there are no changes it is F1
37
between visits
Neuro NA NA 3.5 ND 10 11
designated as conB.
INTRODUCTION !
§ HIV-1 quasispecies contain sequence changes throughout the viral genome. "
2nd round PCR
Tat
Time (mnths)
between visits
V to H
9 10 9 5 18 5
§ Several HIV-1 genes including tat, env, vpr, and nef, as well as the viral LTR, have Patient 107 acquires R2
41
Neuro
Tat
NA NA
V to F
4.5 8 5.5 ND ND
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 neurocognitive impairment Exon
1
Exon
2
51
Time (mnths)
between visits
Neuro norm
23
9
pre-HAART era, single nucleotide changes were observed within the NF-κB-proximal A B
C/EBP and Sp transcription factor binding sites at greater frequency in LTRs isolated Figure 7. Two step PCR to amplify full-length HIV-1 Tat. Step 1: PCR the two exons Tat conB
. . individually using the 4.4kb fragment from clones as template and primers F1 and R1 Time (mnths)
19 8 12 18
from HIV-1-infected patients with more severe disease than those from individuals between visits
with less severe HIV disease. " for exon 1, primers F2 and R2 for exon 2 (R1 and F2 overlap by 17nt). Step 2:, ligate 56
Neuro sub 6 9 9
the two PCR products together using the the PCR products from step 1 and primers F1 Tat V to C
§ These LTRs contained specific single nucleotide polymorphisms (SNPs) in C/EBP
and R2. Time (mnths)
14 2 11 6 3 5
site I (a C-to-T change at position 3 of a consensus B C/EBP binding site I: 3T) that 107
between visits
Neuro norm 12 8 9.5 10 11.5 10.5
were more frequent in tissues derived from individuals who had died with HAD than
Tat V to K V to K
those who died without HAD. "
HIV-1-infected patient 107 Tat
Time (mnths)
36
§ It has been reported that specific LTR SNPs within C/EBP site I (3T) and Sp site III 119
between visits
Neuro norm 5
(5T) increase in prevalence coordinately over the course of HIV-1 infection and
correlate with disease severity." transactivates its co-linear patient- Tat
Time (mnths)
conB
§ 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
Figure 4. (A) The mini bedside neurological exam was modified from
the International HIV Dementia Scale. This test screens for short-term
derived LTR better than an unmatched 131
between visits
Neuro 4
Tat protein in Jurkat T cells
Tat V to I
within susceptible cells of the immune and central nervous systems (CNS). " memory as well as for concentration and processing speed. (B) Patient Time (mnths)
between visits
§ HIV-1-infected monocytes have been shown to be involved in transporting HIV-1 107 was assessed at each visit for neurological impairment. Scores for 220
Neuro 11
across the blood-brain barrier into the CNS where perivascular macrophages and each visit are presented along with the cube drawn at each visit Tat 3 conB
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
Patient-derived HIV-1 LTRs containing Figure 10: Sequence analysis of Tat (diagram of protein depicted above) from several
patients within the DREXELMED HIV/AIDS Genetic Analysis Cohort revealed variation
defining the cellular and viral factors involved in regulating viral transcription in a the 3T CV/EBP site I and 5T Sp site III at position 100 may precede neurocognitive impairment.
number of cellular phenotypes targeted by HIV-1 during the course of infection. "
exhibit a broad spectrum of basal and
HIV-1 LTR structure Tat-mediated transcription activity in
P-TEFb
Jurkat T cells and U-937 monocytic cells CONCLUSIONS!
TAR
TAT
Pol II
Figure 8. LAI or HIV-1-infected patient 107-derived LTR expression construct (1000 ng) ● Previous studies in the pre-HAART era have identified the 3T C/EBP site I and 5T
TBP and expression vectors (500 ng) containing IIIB Tat101, HIV-1-infected patient 107-R00- Sp site III to correlate with the severity of HIV disease or be predictive of those
Nuc-0 Nuc-1
- 405
+ 165
Tat, or 107-R01-Tat were co-transfected into Jurkat T cells. LTR basal and Tat- HIV-1-infected patients more prone to the development of HIV-1-associated
ATF/CREB
neurological disease
Oct-1
mediated transcriptional activity was determined 24 h post-transfection by the Dual-
AP-1
C/EBP
Sp1
C/EBP II
C/EBP I
USF
AP-3
Ets
Lef-1
TATA
NF-κB
Luciferase reporter assay; results were presented as the average fold over LAI basal ● Patient 107 showed the 3T and/or 5T SNPs could be identified prior to the onset of
activity. Representative results shown are derived from transfection experiments disease progression as observed with the decrease in CD4+ T cell count in R03
performed in triplicate.Each experimental result is presented as a mean value (+SD). An (compare Figures 2 and 3).
asterisk (*) denotes a statistically significant difference in activity compared to Tat-
transactivated LAI LTR with P<0.05. Double asterisks (**) denote a statistically ● In addition, the presence of the 3T and 5T SNPs occurred prior to the decrease in
significant difference in activity compared to Tat-transactivated LAI LTR with P<0.01. A neurocognitive performance in the mini-neurological examination.
space indicates nonconsensus changes among the compared residues. ● The results presented demonstrate that different patterns of basal transcription were
Figure 1. The LTR regulates HIV-1 viral gene expression via its interaction with multiple observed in the two cell lines, suggesting that cellular phenotype may alter the
viral and host factors. Genetic variation within the binding sites for these factors has functional properties of 5T- and/or 3T/5T-containing, patient-derived HIV-1 LTRs.
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
Patient-derived HIV-1 Tat from patient ● In addition, the viral transactivator protein Tat contained genetic variations that
DREXELMED cohort study is indicated. 107 have nonconsensus variations ●
demonstrated increased LTR transactivation with its co-linear LTR.
Tat genetic variation at position 100 may be predictive of neurocognitive impairment
Disease severity of patient 107 compared with IIIB or conB Tat
increases over the four visits
Current Number of
Patient Yrs Nadir Current Peak Viral HAART Admitted drugs Drugs tested
Age Viral AIDS-defining
Visit Seropositive CD4 CD4 Load status of abuse status positive for
Load illnesses Figure 5. Patient-derived LTR expression constructs (1000 ng) plus and minus HIV-1
R00 95
(visit 1)
R01
(visit 2)
43
44
3
4
(162)
95
(162)
565
659
455,000
455,000
76
140
0
1
cH
cH
deny any use
admit past use
test negative
test negative
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 FUTURE DIRECTIONS!
with luciferase reporter constructs driven by the HIV-1 LAI LTR and the LAI LTR
R02
(visit 3)
44 5
95
(162)
720 455,000 64 1 cH admit past use test negative
containing the 5T variant. At 24 h post-transfection, LTR basal and Tat-transactivated § Determine if visits R02 and R03 contain alterations in amino acid sequence that
R03 transcription activity was detected by the Dual-Luciferase reporter assay; results were effect transactivation ability."
45 6 95 95 455,000 154 1 dH admit past use test ND
(visit 4)
Figure 9. HIV-1-infected, patient-derived Tat clones from patient 107 were obtained
Figure 2. Clinical parameters for patient 107 were collected. Age is presented in years;
presented as the average fold over LAI basal activity in three independent experiments.
from the 4.4-kb cloned fragments with their co-linear LTRs. Sequence alignments of § Patient 107 has now returned for their R07 visit. Determine the LTR and Tat
Patient-derived HIV-1 LTRs containing the 5T Sp site III induced by IIIB Tat presented sequence from the additional longitudinal visits to analyze for genetic variations that
nadir and current CD4+ T cell count in cells/µl; peak and current viral load in RNA as the average fold over their own basal activity are presented above each graph. 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 correlate to clinical disease progression and LTR and Tat functional alteration."
copies/ml; and HAART status as continuous (cH) or discontinuous (dH). The nadir Results shown are derived from transfection performed in triplicate. Mean values (+SD)
CD4+ T cell count presented in parentheses was the nadir for the patient at all visits for each experimental result are shown. Asterisk (*) denotes a statistically significant the conB Tat. A dot (.) indicates conservation of weak groups. A colon (:) indicates
until R03 where it dropped to 95 cells/µl. ND = not determined. difference in activity compared to the LAI LTR basal activity with P<0.01. conservation of strong groups.
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 "
"