Download Defective HIV-1 Proviruses Can Be Transcribed Upon Activation

Defective HIV-1 Proviruses Can Be Transcribed Upon Activation
1
Ho ,
Ya-Chi
1
Pollack ,
Ross
2
Yong ,
Patrick
Robert F.
1,3
Siliciano
1Department
of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
2Yale University, New Haven, Connecticut; 3Howard Hughes Medical Institute, Baltimore, Maryland
Email: [email protected]
#392
Day 5
(21)
(23)
(13)
+CTL
Day 0
Day 5
78
(10)
(19)
(12)
+CTL
80
(21)
(23)
(13)
Mutated gag DNA
0
1
2
3
4
1000
100
10
1
5
100000000
2
3
4
1000000
100000
10000
1000
100
10
1
82
4
1000
100
10
1
0
5
85
1
2
3
4
gag RNA/gag DNA
100
10
1
3
4
Duration after activation (days)
3
4
100
10
1
0.1
p = 0.048
78
79
80
81
82
100
84
10
85
Median
1
0
1
2
3
4
p = 0.03
p = 0.004
2
3
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78
81
100
82
10
84
85
1
Duration after activation (days)
Hypermutated
AAAAAAA
Large deletion
T cell
activation
AAAAAAA
AAAAAAA
AAAAAAA
Median
Day 1
2
3
4
5
CTL
responses
AAAAAAA
AAAAAAA
AAAAAAA
HIV-1
DNA
Trizol simultaneous DNA/RNA extraction
DNA
 HIV-1 full-length sequencing
 qPCR (gag, RNaseP)
 gag targeted deep sequencing
RNA
 DNase treatment, cDNA synthesis
 qPCR (gag)
 gag targeted deep sequencing
 HIV-1 may not be “transcriptionally silent” in
resting memory CD4+ T cells.
 Low levels of HIV-1 RNA may be transcribed
from both intact and defective HIV-1
proviruses in resting CD4+ T cells.
 HIV-1 DNA quantity per million cells stays unchanged:
cells proliferate upon activation, but there is no significant
preferential proliferation of HIV-1 containing cells.
 A small number of induced proviruses produce the
majority of RNA, but defective proviruses also make a
minority of RNA
85
Median
Median
60%
40%
20%
NON-mutated gag DNA
No CTL
200%
78
80
82
100%
80%
60%
40%
20%
85
No CTL
NON-mutated gag RNA
100%
78
79
80%
80
81
60%
82
40%
84
85
20%
Median
No CTL
+CTL
NON-Mutated gag RNA/gag DNA
Mutated gag RNA/gag DNA DNA
100%
100%
100%
80%
60%
40%
20%
80%
60%
40%
20%
+CTL
78
80%
79
80
60%
81
82
40%
84
20%
85
Median
0%
0%
No CTL
+CTL
p = 0.025
p = 0.025
120%
+CTL
0%
No CTL
+CTL
Median
0%
0%
0%
84
50%
Mutated gag RNA
120%
81
100%
+CTL
140%
79
150%
No CTL
+CTL
No CTL
+CTL
Duration after activation (days)
Day 5
Day 3
80%
0%
Induced
Day 0
84
p = 0.03
80
1
85
0%
Total gag RNA/gag DNA
Figure 3. Changes of HIV-1 DNA and RNA quantities upon CD3/CD28 costimulation. The quantity of cells was calculated by RNaseP copy
numbers. p value was calculated by two-tailed Wilcoxon rank sum test.
HIV-1
RNA
100%
No CTL
79
0
84
100%
+CTL
120%
5
1000
5
82
200%
Total gag RNA
1000
81
82
300%
0%
No CTL
0.1
1
50%
5
10000
10000
1000
0
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2
100000
0.01
2
100%
NON-mutated gag RNA/gag DNA
10000
1000
1
1000000
5
p = 0.01
p = 0.02
p = 0.003
100000
150%
Median
0
Mutated gag RNA/gag DNA
p = 0.025
1
84
100
10000000
10000
p = 0.035
p = 0.004
0
81
1000
200%
NON-mutated gag RNA
100000
3
79
80
5
gag RNA/million cells
10000000
gag RNA/gag DNA
1
p = 0.02
1000000
2
78
Mutated gag RNA
Total gag RNA
1
100000
10000
81
Mutated gag DNA
Total gag DNA
10
0
80
gag DNA/million cells
fold change
1
0.1
Activate resting CD4+ T cells using antiCD3/CD28 costimulation under enfuvirtide
79
80
gag RNA/million cells
fold change
10
10000
Remove
CTLs by
positive
selection
78
79
gag RNA/gag DNA
fold change
100
Isolate CTLs by
negative selection
CD4 activation for 3 days
78
Figure 2. Proportion of HIV-1 DNA and RNA containing inactivating G-to-A mutations upon anti-CD3/CD28 stimulation (day 0, 1, 3, 5) and
CTL co-cultulre.
gag DNA/million cells
fold change
1000
CD4-CTL coculture
for 2 days
Isolate resting
CD4+ T cells
0%
gag RNA/million cells
fold change
10000
10000
Median
20%
NON-mutated gag DNA
gag DNA/million cells
gag DNA/million cells
100000
85
40%
gag RNA/gag DNA
fold change
Total gag DNA
84
60%
(12)
Figure 1. Composition (A) and quantity (B) changes of clonal full-length HIV-1 proviruses upon anti-CD3/CD28 costimulation (day 0 and day 5)
and CTL coculture in two patients (patient 78 and 80). Number in brackets: numbers of clones analyzed.
Total gag RNA/gag DNA
CTL activation for 6 days
Day 0
82
RNA
Patient ID
0
PBMC from HIV-1 infected
individuals under suppressive
antiretroviral therapy
+CTL
80
(10)
(19)
Methods
Activate CTLs using group M consensus
Gag peptide mixture in PBMC for 6 days
Day 5
gag RNA/gag DNA
Conclusions. Hypermutated HIV-1 proviruses can be transcribed both in resting CD4 T cells and upon T cell activation, which may cause
an overestimation of latency reversal of intact proviruses using cell-associated RNA measurements. We propose that CTLs eliminate only a
small number of cells which produce significant amount of HIV-1 RNA, presumably from cells containing intact LTR. The RNA level
decreases after CTL co-culture but the DNA level remains unchanged because the fraction of cells eliminated may be too small compared
with the large quantities of integrated HIV-1 proviruses.
Day 0
gag RNA/million cells
+
# of clones
gag DNA/million cells
Results. The effect of T cell activation on defective HIV-1 proviruses. We found that around 20% of the HIV-1 DNA and around 10% HIV-1
RNA contain inactivating mutations in the gag region, from both resting and activated CD4+ T cells. Upon activation, the quantity of gag DNA
per million cells remains unchanged, while the quantity of gag RNA per million cells increases significantly. The increase of HIV-1 RNA is
most prominent when HIV-1 RNA quantity is normalized to HIV-1 DNA quantity (p <0.05). Using deep sequencing of the full-length gag
genome to quantify the proportion of hypermutated defective sequences, we found that the quantity of hypermutated and nonhypermutated HIV-1 DNA remains unchanged. Both hypermutated and non-hypermutated HIV-1 RNA increases significantly (p <0.05). This
indicates that defective HIV-1 can be transcribed, both at resting state and upon activation.
The effect of CTLs on defective HIV-1 proviruses. Addition of CTLs does not decrease the quantity of hypermutated or non-hypermutated
HIV-1 proviruses. However, both hypermutated and non-hypermunated HIV-1 RNA decreases significantly after CTL co-culture. The quantity
of non-hypermutated HIV-1 RNA decreased (~1 log reduction) more than that of the hypermutated HIV-1 RNA (~0.5 log reduction).
+CTL
78
gag RNA/million cells
Methods. To understand how T cell activation affects the transcription of HIV-1 proviruses, resting CD4+ T cells from aviremic patients under
suppressive antiretroviral therapy were activated with anti-CD3/CD28 costimulation under enfuvirtide to prevent new rounds of in vitro
infection. To examine whether cells containing intact or defective HIV-1 can be eliminated by CTLs, peripheral blood mononuclear cells
(PBMC) containing autologous CTLs were stimulated with group M consensus Gag peptide mixture in the presence of interleukin-2. After 3
days of CD4+ T cell activation and 6 days of CTL activation, pre-stimulated autologous CTLs (magnetic purified) and activated CD4+ T cells
were co-cultured at 1:1 ratio. CTLs were removed by magnetic bead depletion from the CTL-CD4 coculture before simultaneous DNA and
RNA extraction by Trizol reagent. Cell-associated RNA and proviral DNA from cells which are activated for 0, 1, 3, and 5 days and cocultured with CTLs for 2 days was subjected to quantitative PCR and deep-sequencing of the gag region. The full-length gag contains the
start codon and the 9 TGG sequences encoding tryptophan residues. These sites are hotspots APOBEC-mediated G-to-A hypermutations.
Any G-to-A mutation in these sites will lead to either a missense methionine-to-isoleucine change of the start codon or a nonsense mutation
of the tryptophan residues into premature stop codons, and render the provirus defective.
Day 5
81
Day 0
Day 1
Day 3
Day 5
+CTL
Day 0
80
Day 0
Day 1
Day 3
Day 5
+CTL
1
79
Day 0
Day 1
Day 3
Day 5
+CTL
0%
78
80%
gag DNA/million cells
fold change
20%
10
0%
gag RNA/million cells
fold change
Hypothesis. We hypothesize that some defective proviruses can be transcribed and translated upon T cell activation, but only a minority of
them can be eliminated by CTLs. Understanding of the dynamics of defective HIV-1 transcription upon activation and elimination by CTLs
can facilitate the correct measurement of the efficacy of latency reversing agents.
Intact
Point mutation
Packaging signal deletion
Hypermut
Deletion
gag RNA /gag DNA
fold change
40%
100
20%
Day 0
Day 1
Day 3
Day 5
+CTL
60%
40%
Day 0
Day 1
Day 3
Day 5
+CTL
80%
60%
Day 0
Day 1
Day 3
Day 5
+CTL
1000
100%
DNA
Day 0
Day 1
Day 3
Day 5
+CTL
B
80%
Day 0
Day 1
Day 3
Day 5
+CTL
A
HIV-1 DNA /million cells
Background. Human immunodeficiency virus type-1 (HIV-1) persists in the latent reservoir, primarily resting memory CD4+ T cells, as the
major barrier to cure. HIV-1 proviruses reside in the resting memory CD4+ T cells as three types: induced proviruses, intact noninduced
proviruses and defective proviruses. The defective provirus is comprised of ~32% of APOBEC-mediated G-to-A hypermutations, ~46% of
large internal deletions, ~6% of packaging signal deletions/mutations, and ~4% of inactivating point mutations. Since these defective
proviruses lack epigenetic silencing and may have intact long terminal repeat (LTR) promoter function, it is possible that they can be
transcribed and translated upon activation, such as by antigen stimulation, by homeostatic proliferation and by latency reversing agents
used in the shock-and-kill strategy for HIV-1 eradication. Transcription of defective proviruses may complicate the correct estimation of the
efficacy of latency reversal of inducible proviruses using cell-associated RNA measurements. Whether cells containing defective proviruses
can be eliminated by cytotoxic T lymphocytes (CTLs) upon activation remains unknown.
%gag containing inactivating G-to-A mutations
Results
Abstract
 CTLs may eliminate a small number of cells which
produce the majority of HIV-1 RNA. Therefore, the total
HIV-1 DNA quantity does not decrease, as the
proportion is too small to make significant changes..
 The decrease of hypermutated HIV-1 RNA suggests
that cells containing defective proviruses may be
recognized and eliminated by CTLs.
Figure 5. Proposed dynamics of defective HIV-1 proviruses upon T cell activation and CTL elimination.
Figure 4. Changes of HIV-1 DNA and RNA quantities upon CTL co-culture. The quantity of cells was calculated by RNaseP copy
numbers. p value was calculated by two-tailed Wilcoxon rank sum test.
Conclusions
1. The effect of T cell activation on hypermuted/non-hypermuted HIV-1:
1) HIV-1 DNA level (copies per million cells, as measured by gag DNA/RNaseP) remains unchanged in cells containing hypermuted and non-hypermuted proviruses.
2) HIV-1 RNA level (copies per million cells, as measured by gag RNA/RNaseP) increases in both hypermuted and non-hypermuted samples, indicating that even
hypermutated samples can be transcribed upon T cell activation. This effect is most prominent when measured as gag RNA copies/gag DNA copies, which reflects HIV-1
RNA reactivation from comparable numbers of HIV-1 proviruses in each sample.
2. The effect of CTL on cells containing hypermutated/non-hypermutated HIV-1:
1) HIV-1 DNA level (copies per million cells) remains unchanged in cells containing hypermut and non-hypermut proviruses.
2) HIV-1 RNA level (copies per million cells) decreases in hypermutated gag RNA (~3-fold reduction) and non-hypermutated gag RNA (~10-fold reduction).
3. We propose that CTLs eliminate only a small number of cells which produce significant amount of HIV-1 RNA. The fraction of cells eliminated may be too small in proportion
to all the integrated HIV-1 DNA. Therefore, RNA level decreases but DNA levels remain unchanged. It cannot be distinguished thus far whether it is the induced, intact
noninduced, or defective proviruses with large internal deletions which produce the non-hypermutated RNA.
Acknowledgements
We thank all study participants. We thank Dr. Joel N. Blankson for his critical advice, Adam Longwich for study subject recruitment and coordination, and Dr. Haiping Hao for
deep sequencing. This work was supported by the Martin Delaney CARE and DARE Collaboratories (NIH grants AI096113 and 1U19AI096109), by an ARCHE Collaborative
Research Grant from the Foundation for AIDS Research (amFAR 108165-50-RGRL), by the Johns Hopkins Center for AIDS Research (P30AI094189), by NIH grant 43222, by
the Howard Hughes Medical Institute and by the Bill and Melinda Gates Foundation.