Isolation of a cellular factor that can reactivate latent HIV-1 without T cell activation.
ABSTRACT HIV-1 latency in resting CD4(+) T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). Eliminating the latent HIV-1 reservoir may require the reactivation of viral gene expression in latently infected cells. Most approaches for reactivating latent HIV-1 require nonspecific T cell activation, which has potential toxicity. To identify factors for reactivating latent HIV-1 without inducing global T cell activation, we performed a previously undescribed unbiased screen for genes that could activate transcription from the HIV-1 LTR in an NF-kappaB-independent manner, and isolated an alternatively spliced form of the transcription factor Ets-1, DeltaVII-Ets-1. DeltaVII-Ets-1 activated HIV-1 transcription through 2 conserved regions in the LTR, and reactivated latent HIV-1 in cells from patients on HAART without causing significant T cell activation. Our results highlight the therapeutic potential of cellular factors for the reactivation of latent HIV-1 and provide an efficient approach for their identification.
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ABSTRACT: Antiretroviral therapy fails to cure HIV-1 infection because latent proviruses persist in resting CD4(+) T cells. T cell activation reverses latency, but <1% of proviruses are induced to release infectious virus after maximum in vitro activation. The noninduced proviruses are generally considered defective but have not been characterized. Analysis of 213 noninduced proviral clones from treated patients showed 88.3% with identifiable defects but 11.7% with intact genomes and normal long terminal repeat (LTR) function. Using direct sequencing and genome synthesis, we reconstructed full-length intact noninduced proviral clones and demonstrated growth kinetics comparable to reconstructed induced proviruses from the same patients. Noninduced proviruses have unmethylated promoters and are integrated into active transcription units. Thus, it cannot be excluded that they may become activated in vivo. The identification of replication-competent noninduced proviruses indicates that the size of the latent reservoir-and, hence, the barrier to cure-may be up to 60-fold greater than previously estimated. PAPERFLICK:Cell 10/2013; 155(3):540-51. · 31.96 Impact Factor
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ABSTRACT: Highly active antiretroviral therapy can suppress plasma viral loads of HIV-1 infected individuals to below the detection limit of standard clinical assays. However, low-level viremia still persists. Many patients also have transient viral load measurements above the detection limit (the so-called "viral blips"). The latent reservoir consisting of latently infected CD4+ T cells represents a major obstacle to HIV-1 eradication. These cells can be activated to produce virions but the size of the latent reservoir is relatively stable. The mechanisms underlying low viral load persistence, emergence of intermittent viral blips and stability of the latent reservoir are not well understood. Cellular and viral transcription factors play an important role in the establishment and maintenance of HIV-1 latency. Infected cells with intermediate transcriptional activities may either revert to a latent state or become highly activated and produce virions due to intracellular perturbations. Here we develop a mathematical model that includes such stochastic population switch. We demonstrate that the model can generate a stable latent reservoir, intermittent viral blips, as well as low-level viremia persistence. Latently infected cells with intermediate transcription activities may maintain their size through a high level of homeostatic proliferation, while cells with low transcriptional activities are likely to be maintained through the reversion from cells with intermediate transcription activities. Simulations also suggest that treatment intensification or activation therapy may not help to eradicate the latent reservoir. Blocking the proliferation of latently infected cells might be a good strategy. These results provide more insights into the long-term dynamics of virus and latently infected cells in HIV patients on suppressive therapy and may help to develop novel treatment strategies.Journal of Theoretical Biology 07/2014; · 2.35 Impact Factor
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ABSTRACT: Attempts to eradicate HIV have been thwarted by the persistence of a small pool of quiescent memory CD4 T cells that harbor a transcriptionally silent, integrated form of the virus that can produce infectious virions following an anamnestic immune response. Transcription factors downstream of T-cell receptor activation, such as NF-κB/Rel and nuclear factor of activated T cells (NFAT) transcription members, are considered important regulators of HIV transcription during acute HIV infection. We now report studies exploring their precise role as antagonists of HIV latency using cell and primary CD4 T cell models of HIV-1 latency. Surprisingly, RNA interference studies performed in J-Lat CD4 T cells suggested that none of the NFATs, including NFATc1, NFATc2, NFATc3, and NFAT5, played a key role in the reactivation of latent HIV. However, cyclosporin A markedly inhibited the reactivation response. These results were reconciled when calcium signaling through calcineurin was shown to potentiate prostratin induced activation of NF-κB that in turn stimulated the latent HIV long terminal repeat (LTR). Similar effects of calcineurin were confirmed in a primary CD4 T cell model of HIV latency. These findings highlight an important role for calcineurin in NF-κB-dependent induction of latent HIV transcription. Innovative approaches exploiting the synergistic actions of calcineurin and prostratin in the absence of generalized T-cell activation merit exploration as a means to attack the latent viral reservoir.PLoS ONE 10/2013; 8(10):e77749. · 3.53 Impact Factor
Isolation of a cellular factor that can reactivate latent
HIV-1 without T cell activation
Hung-Chih Yanga, Lin Shena, Robert F. Silicianoa,b, and Joel L. Pomerantzc,d,1
Departments ofaMedicine andcBiological Chemistry,dInstitute for Cell Engineering, andbHoward Hughes Medical Institute, Johns Hopkins University
School of Medicine, Baltimore, MD 21205
Edited by Diane E. Griffin, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, and approved February 6, 2009 (received for review
September 23, 2008)
HIV-1 latency in resting CD4?T cells represents a major barrier to
virus eradication in patients on highly active antiretroviral therapy
(HAART). Eliminating the latent HIV-1 reservoir may require the
reactivation of viral gene expression in latently infected cells. Most
approaches for reactivating latent HIV-1 require nonspecific T cell
activation, which has potential toxicity. To identify factors for
reactivating latent HIV-1 without inducing global T cell activation,
we performed a previously undescribed unbiased screen for genes
that could activate transcription from the HIV-1 LTR in an NF-?B-
independent manner, and isolated an alternatively spliced form of
the transcription factor Ets-1, ?VII-Ets-1. ?VII-Ets-1 activated HIV-1
transcription through 2 conserved regions in the LTR, and reacti-
vated latent HIV-1 in cells from patients on HAART without causing
significant T cell activation. Our results highlight the therapeutic
potential of cellular factors for the reactivation of latent HIV-1 and
provide an efficient approach for their identification.
antiretroviral therapy ? ?VII-Ets-1 ? expression cloning ?
long terminal repeat ? viral reservoir
ever, there is still no therapeutic regimen to cure chronic HIV-1
infection. Although highly active antiretroviral therapy
(HAART) can suppress plasma viral load to undetectable levels,
viremia rebounds within weeks after discontinuation of
HAART. The major barrier to eradication of HIV-1 infection is
the existence of viral reservoirs. Among them, the best charac-
terized is a small pool of latently-infected resting memory CD4?
T cells harboring an integrated provirus (2–4). Previous studies
on HAART (5). The half-life of this reservoir was estimated to
be ?44 months. At this rate of decay, it is expected to take ?60
years to purge HIV-1 from infected patients on HAART. Thus,
this reservoir necessitates the lifetime use of HAART, and
strategies are needed for eradication of latently infected
cells (6, 7).
Recently, reactivation of latent virus has gained wide interest
as a potential strategy to eradicate the viral reservoirs (8–11). It
is assumed that latently infected cells can be killed either by
immune attack or direct viral cytopathic effects after reactiva-
tion of latent HIV-1. A reactivation strategy, along with simul-
taneous efficient suppression of viral spread by HAART, might
reduce and ultimately eliminate the latent reservoirs (6, 7).
Although logical, this approach has practical limitations. Be-
cause signals that cause T cell activation also activate HIV-1
replication, some studies have focused on strategies to induce
some level of T cell activation as a means of reactivating latent
HIV-1 (10, 11). Unfortunately, the potential toxicity of such
nonspecific T cell activation has severely complicated this ap-
proach (10, 11). For example, patients treated with agonistic
anti-CD3 monoclonal antibody and IL-2 suffered from severe
side effects, transient renal failure, and seizure. An ideal reac-
tivation strategy for virus eradication might allow activation of
HIV-1 without inducing global T cell activation.
dvances in antiretroviral therapy have dramatically reduced
mortality among patients with HIV-1 infection (1). How-
The HIV-1 provirus responds to various extracellular stimuli,
including T cell activation signals and some proinflammatory
cytokines (12–14). The HIV-1 promoter, located within the U3
region of the LTR, contains an array of cis-acting transcription
factor binding sites (15). The interaction between these diverse
signals and the various binding sites in the LTR forms a complex
regulatory network. In particular, the host transcription factor
NF-?B is important for activating HIV-1 gene expression
through 2 conserved ?B sites in the core enhancer region of the
HIV-1 LTR (12, 13). However, HIV-1 can replicate in the
absence of ?B sites in the LTR (16), consistent with the existence
of NF-?B-independent pathways in the activation of HIV-1 (17,
18). NF-?B also has a critical role in innate and adaptive immune
responses, and regulates genes that have important roles during
T cell activation (19). Because of the central role of NF-?B in T
cell activation, we reasoned that to find genes that could
uncouple the activation of latent HIV-1 from T cell activation it
would be desirable to identify factors that could activate the
HIV-1 LTR in an NF-?B-independent manner.
To systematically search for NF-?B-independent pathways for
the activation of HIV-1, we performed an expression cloning
screen using a reporter containing mutated NF-?B sites in the
enhancer region of the HIV-1 LTR. By screening a human
splenocyte cDNA expression library, we isolated an alternatively
spliced form of the Ets-1 transcription factor, ?VII-Ets-1. ?VII-
Ets-1 was able to activate the NF-?B site-mutated HIV-1 LTR
without stimulating T cell activation and could activate latent
HIV-1 from resting CD4?T cells isolated from patients on
potential of this expression cloning strategy to yield novel
approaches for eradicating latent reservoirs of HIV-1.
Expression Cloning Screen to Identify NF-?B-Independent Pathways
for the Reactivation of Latent HIV-1. To facilitate the identification
of NF-?B-independent pathways that could activate the HIV-1
LTR, we generated a luciferase reporter, m?B-LTR-Luc, which
contains the HIV-1 LTR from reference strain NL4-3 with
mutated ?B sites within the core enhancer region (?106 to ?83)
(Fig. 1A) that have been shown to abolish the activity of NF-?B
on the HIV-1 LTR (13). We then screened a human splenocyte
cDNA expression library for the ability to stimulate the m?B-
LTR-Luc reporter. To maximize the number of the cDNAs that
could be assayed, we generated cDNA pools with ?100 cDNAs
Author contributions: H.-C.Y., R.F.S., and J.L.P. designed research; H.-C.Y. and L.S. per-
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
April 14, 2009 ?
vol. 106 ?
no. 15 ?
per pool (20). Each pool was transfected into HEK293T cells
with the m?B-LTR-Luc reporter and pCSK-lacZ, an NF-?B-
independent ?-galactosidase expression vector used for normal-
izing transfection efficiency and extract recovery. A pool was
considered positive if it activated the m?B-LTR-Luc reporter by
3-fold or more, as compared with an empty expression vector. In
all, 477 pools were screened (Fig. 1B), and 7 positive pools were
To verify that each positive pool activated the HIV-1 LTR in
an NF-?B-independent manner, we tested each pool for its
ability to activate the NF-?B-responsive reporter Ig?2-IFN-GL4,
which contains 2 NF-?B sites upstream of the IFN-? minimal
promoter and the luciferase gene (20). Four of the seven positive
pools did not activate the Ig?2-IFN-GL4 reporter. For example,
although pool 50 activated the m?B-LTR-Luc reporter by 3.4-
fold, pool 50 did not activate the Ig?2-IFN-GL4 reporter at all
(Fig. 1C). We purified the activity of 1 pool (pool 50) for further
Isolation of Human ?VII-Ets-1 from a Human Splenocyte cDNA Library.
To identify the cDNA responsible for the activity of pool 50,
DNA from pool 50 was used to retransform bacteria, and
individual colonies were amplified in wells of 24-well plates.
Each 24-well plate was screened as a subpool after preparing
DNA from pooled aliquots from each well. Once a 24-well plate
was found to exhibit the activity present in the original pool 50,
a conceptual matrix (4 row ? 6 column) was used to identify
the coordinates of the positive clone within the 24-well plate
(Fig. 1D). Sequencing revealed that this clone was a previously
described spliced variant of the transcription factor Ets-1,
Ets-1 is a member of Ets family of transcription factors that
(21, 22). Ets-1 is highly expressed in T cells, and has an essential
role in their proliferation and survival (23). Two isoforms of
Ets-1, full-length (fl)-Ets-1 and ?VII-Ets-1, which lacks the
region encoded by exon VII, can be detected in T cells; however,
fl-Ets-1 is expressed at a much higher level (21, 22). The
functional domains of Ets-1 are shown in Fig. 1E. Previous
studies have revealed that exon VII encodes an autoinhibitory
domain that regulates the DNA binding activity of Ets-1 (24).
The C-terminal portion of the exon VII-encoding region con-
tains helices that inhibit the DNA-binding domain in concert
with helices encoded C-terminal to the DNA-binding domain of
Ets-1 (ETS). The N-terminal portion of the exon VII domain is
phosphorylated in a calcium-responsive manner, and phosphor-
ylation in this region further inhibits DNA binding and tran-
scriptional activation (25). Because ?VII-Ets-1 lacks the exon
VII-encoded region, its DNA-binding affinity is significantly
enhanced. The role of fl-Ets-1 on HIV-1 gene expression has
been studied before (26, 27). Full-length Ets-1 binds upstream to
the core enhancer of HIV-1 LTR, and stimulates viral gene
expression by interacting with other transcription factors, such as
NF-?B, NFAT, and USF-1. Because HIV-1 replication is closely
linked to the activation state of the T cell, we determined the
expression of fl-Ets-1 and ?VII-Ets-1 in resting and activated
CD4?T cells. Both fl-Ets-1 and ?VII-Ets-1 were readily de-
tectable in resting and activated CD4?T cells; however, the
expression of ?VII-Ets-1 was less than that of fl-Ets-1 (Fig. 1F).
Specific Activation of the HIV-1 LTR by ?VII-Ets-1. We verified that
?VII-Ets-1 was responsible for the activity displayed by pool 50.
Overexpression of ?VII-Ets-1 in HEK293T cells activated the
wild-type HIV-1 LTR (wt-LTR-Luc) and the LTR with mutated
NF-?B sites (m?B-LTR-Luc), but did not activate the NF-?B-
dependent reporter (Ig?2-IFN-GL4) (Fig. 2A). Thus, ?VII-Ets-1
was able to specifically activate the HIV-1 LTR without acti-
vating NF-?B. Compared with its effect on m?B-LTR-Luc,
?VII-Ets-1 showed a higher level of induction on wt-LTR-Luc,
suggesting that, although ?VII-Ets-1 did not require NF-?B for
transcriptional activity, it does act cooperatively with NF-?B in
the context of the HIV-1 LTR. This finding is consistent with a
previous report demonstrating the cooperative activity between
Ets-1 and NF-?B on the HIV-1 LTR (26). When directly
site directed mutagenesis
2 X2 X????
3 X Sp13 X Sp1
Ig Igκ κ2- 2-
m mκ κB-
the 5? LTR and the untranscribed region of Gag (GLS) derived from NL4-3 upstream of the luciferase gene. Two tandem NF-?B sites (2x ?B), 3 Sp1 sites (3x Sp1)
and the TATA box (TATA) in the U3 region are shown as boxes with indicated colors. The NF-?B-binding sites were mutated as indicated. Numbering above the
were transfected into HEK293T cells with the m?B-LTR-Luc reporter and the pCSK-lacZ control vector, and the fold stimulation was determined as described in
Materials and Methods. Only 150 out of 477 pools screened are shown. (C) HEK293T cells were cotransfected with pool 50, Ig?2-IFN-GL4 and pCSK-lacZ. The fold
of subpools. The well in R 1 and C 5 contained the positive clone. (E) The functional domains of fl-Ets-1 and ?VII-Ets-1. The exon VII, originally designated in
alignment with chicken c-ets gene, is actually exon 6 in the human ETS1 gene (22). (F) Western blot of fl-Ets-1 and ?VII-Ets-1 in 10 ?g of lysates of primary resting
for the T cell lysates.
www.pnas.org?cgi?doi?10.1073?pnas.0809536106Yang et al.
compared in this assay, ?VII-Ets-1 showed a greater ability than
fl-Ets-1 to activate the m?B-LTR-Luc reporter at comparable
expression levels (Fig. 2B). To confirm that ?VII-Ets-1 could
activate the HIV-1 LTR in T cells, we also assessed its ability to
activate the m?B-LTR-Luc in the Jurkat T cell line. Overex-
pression of ?VII-Ets-1 in Jurkat T cells did induce the m?B-
LTR-Luc reporter, but to a lower degree than in HEK293T cells
(Fig. 2C). As in HEK293T cells, ?VII-Ets-1 displayed more
activity than fl-Ets-1 in Jurkat T cells (Fig. 2C).
The DNA-Binding Domain of ?VII-Ets-1 Is Essential for Activation of
HIV-1 LTR. ?VII-Ets-1 contains an N-terminal transactivation
domain and C-terminal DNA-binding domain (Fig. 3A). To
investigate the functional domains that are responsible for the
activation of m?B-LTR-Luc, we generated 3 FLAG-tagged
mutants: TAD-?VII-Ets-1 (transactivation domain of ?VII-Ets-
1), DBD-?VII-Ets-1 (DNA-binding domain of ?VII-Ets-1), and
mDBD-?VII-Ets-1 (R304A, R307A mutant of ?VII-Ets-1).
R304 and R307 of ?VII-Ets-1 are equivalent to R391 and R394
of fl-Ets-1. These 2 arginine residues are critical for the DNA
binding of Ets-1 (28). Cotransfection of m?B-LTR-Luc and each
of these variants revealed that TAD-?VII-Ets-1 alone could not
activate the mutant HIV-1 LTR, whereas DBD-?VII-Ets-1
alone had a moderate effect (Fig. 3A). Also, mutation of the 2
essential arginine residues (R304 and R307) in mDBD-?VII-
Ets-1 abolished the stimulatory effect of ?VII-Ets-1, indicating
the necessity of the DBD-?VII-Ets-1 in activation of the HIV-1
LTR (Fig. 3A). The expression of these ?VII-Ets-1 variants in
HEK293T cells was confirmed by Western blotting with anti-
FLAG antibodies (Fig. 3B). These results demonstrate the
essential role of DNA binding in the activation of the HIV-1
LTR by ?VII-Ets-1.
Mapping of the ?VII-Ets-1-Responsive Elements in HIV-1 LTR. We
assayed a panel of LTR mutant and deletion constructs to
determine which DNA elements in the LTR are required for
?VII-Ets-1-mediated activation (Fig. 4A). Previous studies re-
vealed that Ets-1 can bind to the Ets-binding site (EBS) in the
distal region (?150 to ?145) of the LTR (27). Also, there are
50 25 12.5 100
0 0.51 1.52 2.53
5060 7080 90100
?VII-Ets-1 expression vector in the presence of 0.5 ng of the control vector
TK-RLuc plus 1 of 3 different reporters: 4 ng wt-LTR-Luc, 4 ng m?B-LTR-Luc,
or 10 ng Ig?2-IFN-GL4 in HEK293T cells. The fold stimulation is shown
normalized to that observed with the reference control vector pmax-
Empty. Data are means ? SD of triplicate transfections, and represent 2
vectors in the presence of 4 ng m?B-LTR-Luc and 0.5 ng pTK-RLuc in
HEK293T cells. (Upper) Fold stimulation, normalized to that observed with
pmax-Empty. Data are means ? SD of triplicate transfections, and repre-
sent 2 independent experiments. (Lower) Western blot analysis of the
relative expression level of fl-Ets1 and ?VII-Ets-1 using the lysates analyzed
presence of 100 ng m?B-LTR-Luc and 50 ng pTK-RLuc in Jurkat T cells. The
fold stimulation is shown normalized to that observed with pmax-Empty.
Data are means ? SD of triplicate transfections, and represent 2 indepen-
?VII-Ets-1 specifically activates the HIV-1 LTR. (A) Titration of
135 243 244
135 243 244
HEK293T cells with 100 ng of FLAG-tagged ?VII-Ets-1, TAD-?VII-Ets-1, DBD-
?VII-Ets-1, or mDBD-?VII-Ets-1 with 4 ng of m?B-LTR-Luc and 0.5 ng of TK-
RLuc. (Left) Displayed are the constructs assayed. (Right) Fold stimulation is
shown normalized to that observed with pmax-Empty. Data are means ? SD
of triplicate transfections, and represent 3 independent experiments. (B)
Western blot analysis of the relative expression level of FLAG-tagged ?VII-
Ets-1 variants using the identical HEK293T cell lysates analyzed in A.
Mapping the functional domains of ?VII-Ets-1. (A) Cotransfection of
Yang et al.PNAS ?
April 14, 2009 ?
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another 2 EBSs that bind the Ets family transcription factor
GABP? within the HIV-1 core enhancer (?106 to ?83) (29).
These 2 EBSs are located in the 3? half of ?B sites with the core
sequence 5?-GGAA-3? (Fig. 4A). Mutation or deletion of the
EBS in the distal region (?150 to ?145) (mEBS-m?B-LTR-Luc
and delEBS-m?B-LTR-Luc) reduced ?VII-Ets-1-mediated ac-
tivation by ?40%. However, deletion of the enhancer region of
LTR (delEnh-LTR-Luc), which contains binding sites for NF-
?B, NFAT and GABP?, did not significantly reduce ?VII-Ets-
1-mediated activation, either in the context of m?B-LTR-Luc or
mEBS-LTR-Luc, indicating that the 2 defined EBSs in this core
enhancer region are not required for ?VII-Ets-1 activity. A
construct containing only the 3 Sp1 binding sites and the TATA
box (Sp1-TA-LTR-Luc) displayed ?60% of the activity of the
m?B-LTR-Luc. Deletion of the Sp1-binding sites in the context
of Sp1-TA-LTR-Luc destroyed the activity of ?VII-Ets-1, indi-
cating that this region is as important for ?VII-Ets-1 respon-
siveness as is the EBS between ?150 and ?145 (Fig. 4B).
Interestingly, the ?VII-Ets-1 mutant that could not bind DNA
(mDBD-?VII-Ets-1) did not display any activity on any of these
reporter constructs, indicating that the DNA-binding activity of
?VII-Ets-1 is required for transactivation through the elements
between ?150 and ?145 and between ?82 and ?44.
Overexpression of ?VII-Ets-1 Does Not Induce Full Activation of
Primary CD4?T Cells. Because our goal was to find a way to
reactivate latent HIV-1 without inducing global T cell activation,
we next determined whether overexpression of ?VII-Ets-1 could
activate resting CD4?T cells. We isolated resting CD4?T cells
with high purity from HIV-1 negative donors, and then trans-
fected the cells with either a mock vector or the ?VII-Ets-1-
expression vector under conditions that achieved 50–70% trans-
fection of cells. Overexpression of ?VII-Ets-1 in resting CD4?T
cells did not cause the up-regulation of the T cell activation
markers CD69, CD25, or HLA-DR, when compared with the
mock vector. In contrast, very strong up-regulation of these
proteins was observed in cells activated with agonistic anti-CD3
and anti-CD28 antibodies (Fig. 5A). We also compared the
expression of IFN-? and IL-2 in these cells. The cells transfected
with either mock vector or ?VII-Ets-1 had very low levels of
mRNA for IFN-? and IL-2, whereas the transcripts of IFN-? and
IL-2 in PHA-activated cells were significantly up-regulated by
147- and 10-fold, respectively (Fig. 5 B and C). We first assessed
cell proliferation by staining the cells with propidium iodide. A
low fraction of cells transfected with either mock vector or
?VII-Ets-1 had entered S phase (?5%), whereas 34% of cells
costimulated by anti-CD3 and anti-CD28 antibodies entered S
phase (Fig. 5D). To independently assess proliferation, we
stained resting CD4?T cells with CFSE before transfection;
?2% of cells transfected with either mock vector or ?VII-Ets-1
proliferated, whereas almost 18% cells treated with anti-CD3
and anti-CD28 for 3 days proliferated (Fig. 5E). These data
indicate that overexpression of ?VII-Ets-1 does not induce
significant T cell activation.
Ability of ?VII-Ets-1 Overexpression to Stimulate Virus Production by
Resting CD4?T Cells from HIV-1-Infected Patients on HAART. The
results of our transient reporter assay demonstrated that ?VII-
Ets-1 can activate the HIV-1 LTR without the involvement of
NF-?B. We extended this result by determining whether over-
expression of ?VII-Ets-1 could reactivate HIV-1 from the latent
reservoir in patients on HAART. We isolated highly purified
resting CD4?T cells (?99%) from 6 different patients whose
viral loads were below the limit of detection (?50 copies/mL of
HIV-1 RNA), and then transfected these T cells with either a
GFP-expressing or empty expression vector to serve as a nega-
expression vector encoding HIV-1 Tat, a strong transactivator of
the LTR that induces high levels of virus production when
overexpressed in latently infected cells (30, 31).
Overexpression of ?VII-Ets-1 resulted in an increase of virus
release from 9- to 560-fold, indicating that overexpression of
?VII-Ets-1 alone is sufficient to cause virus production in these
resting CD4?T cells of patients on HAART (Table 1). This
stimulation was comparable with that achieved with the positive
control Tat-expression vector. The transfection efficiency was
estimated to be 45 to 70% based on the frequency of GFP? cells
among cells transfected with the GFP-expression vector. For 3
patients, we also tested whether fl-Ets-1 expression could acti-
vate virus production. Full-length Ets-1 expression resulted in a
measurable activation of virus, but the effect was weaker than
that observed with either ?VII-Ets-1 or Tat, consistent with the
weaker ability of fl-Ets-1 to activate transcription from the
HIV-1 LTR (Fig. 2).
The reactivation of latent HIV-1 provirus is an essential pre-
requisite for the eradication of HIV-1 infection. We have
demonstrated that an unbiased screen for activators of LTR-
dependent transcription is a straightforward approach toward
the identification of activities that can reactivate latent HIV-1.
We isolated ?VII-Ets-1 as an activity that was sufficient to
3 X Sp1
R U5 GLS
2 X m??B
R U5 GLS
R U5 GLS
R U5 GLS
R U5 GLS
R U5 GLS
R U5 GLS
Reporter constructs with truncations or point mutations of the HIV-1 LTR.
Numbering indicates the nucleotide positions relative to the transcription
start site (?1). The underlined sequences within the ?B sites indicate putative
of ?VII-Ets-1-expression vector, mDBD-?VII-Ets-1, or pmax-Empty with the
indicated reporters. The fold stimulation normalized to that observed with
pmax-Empty is shown. Data are means ? SD of triplicate transfections, and
represent 2 independent experiments.
Mapping the ?VII-Ets-1-responsive elements in the HIV-1 LTR. (A)
www.pnas.org?cgi?doi?10.1073?pnas.0809536106Yang et al.
induce transcription from the HIV-1 LTR. When introduced
?VII-Ets-1 could activate HIV-1 production without inducing T
The HIV-1 LTR integrates the function of a multitude of
transcription factors, some of which respond to signaling path-
ways that are central to cell activation, proliferation, and sur-
vival. Our results indicate that it is possible to use only one of the
factors that contributes to LTR regulation to activate latent
HIV-1 provirus without the need to activate multiple transcrip-
tion factors or pathways that may be toxic. Other regulators of
To focus our screen on activities that would be less likely to
induce T cell activation, we used a mutant LTR that could not
respond to NF-?B. This approach removed from our analysis the
high frequency of genes that can activate NF-?B when overex-
pressed (20), and targeted the detection of genes that function
through elements of the LTR distinct from the NF-?B-binding
sites. However, it is still possible that some activators of NF-?B
may be particularly efficient at activating latent HIV-1 without
inducing T cell activation.
Full-length Ets-1 has previously been shown to participate in
LTR regulation in concert with other transcription factors, like
NF-?B, NFAT, and USF-1 (26, 27), by binding to a conserved
(32). Our results demonstrate that overexpression of the alter-
natively spliced form of Ets-1, ?VII-Ets-1, is sufficient both for
LTR transcriptional activation and activation of latent HIV-1.
The activation of the LTR by ?VII-Ets-1 required its DNA-
binding activity, and proceeded through 2 conserved regions of
the LTR, the region containing the Ets-1-binding site (?150 to
?145) and the region of the LTR containing the Sp1 binding
sites (3x Sp1) (?82 to ?44). Although Ets-1 has not previously
been reported to bind between ?82 and ?44, it is possible that
?VII-Ets-1 can bind to a previously unidentified EBS in this
region. Alternatively, ?VII-Ets-1 may activate a cellular gene
that in turn acts in this region to induce transcription from the
HIV-1 LTR. Further studies are necessary to resolve these
possibilities. ?VII-Ets-1 is an isoform that is not subject to the
regulation imposed by the autoinhibitory domain that is encoded
by exon VII, or by phosphorylation sites also encoded by exon
VII that negatively regulate Ets-1 DNA binding and transcrip-
tional activity. Our data indicate that ?VII-Ets-1 is a more
potent activator of LTR-dependent transcription than fl-Ets-1,
and this enhanced activity correlates with a greater ability to
activate HIV-1 production. Therefore, the ?VII-Ets-1 isoform
may be uniquely suited to being active without the need for other
factors or pathways that may be required for the activity of
fl-Ets-1. The ability of ?VII-Ets-1 to reactivate latent HIV-1
levels of expression of this isoform of Ets-1 in latently infected
cells. Alternatively, potential therapies might involve the con-
version of fl-Ets-1 from an inhibited to an active transcription
Our screen was conducted with only 1 cDNA expression
library, and screening was well below saturation. Therefore, it is
likely that our approach can be expanded and lead to the
isolation of further interesting genes with desirable therapeutic
possibilities. Our results prove that the screen for activators of
LTR transcription is a good surrogate screen for genes that can
activate latent HIV-1. It will allow efficient, inexpensive screen-
ing of more cDNA libraries, and can be adapted easily for the
screening of small molecules.
0 200400 6008001000
0200 400600 8001000
Mock VII-Ets-1 PHA
?VII-Ets-1-transfected primary resting CD4?T cells or CD4?T cells activated
with 2.5 ?g/mL anti-CD3 and 1 ?g/mL anti-CD28 for 72 h; 5 ?g of ?VII-Ets-1-
expression vector was transfected into 3.5 ? 106primary resting CD4?T cells
by nucleofection. ?VII-Ets-1-transfected cells were analyzed for surface ex-
IFN-? (B) and IL-2 (C) transcripts from primary CD4?T cells 72 h after trans-
fection or after 72 h of PHA treatment were quantified by real-time RT-PCR,
and were normalized to ubiquitin mRNA levels. The fold stimulation was
normalized to that observed with the reference control vector pmax-Empty.
Data are means ? SD of triplicate measurements, and represent 2 indepen-
dent experiments. (D) ?VII-Ets-1-transfected cells, pmax-Empty-transfected
cells, and anti-CD3/anti-CD28-treated cells were stained with propidium io-
dide, and analyzed for cell proliferation by flow cytometry on day 3 after
nucleofection or antibody treatment. (E) Primary CD4?T cells were stained
with 1 ?M CFSE and then transfected with ?VII-Ets-1 expression vector or
pmax-Empty or activated with anti-CD3 and anti-CD28, and analyzed for cell
proliferation by flow cytometry on day 3 after nucleofection or antibody
Overexpression of ?VII-Ets-1 in primary resting CD4?T cells does not
Table 1. Induction of virus production from primary CD4?T cells
isolated from patients on HAART by transfection of ?VII-Ets-1,
fl-Ets-1, or Tat
The number in each cell represents the copy number of viral RNA released
in the culture supernatant. The number in parentheses is the fold increase, as
compared with the reference control vector pmax-GFP (patients 1–3) or
pmaxEmpty (patients 4–6). ND, not determined.
Yang et al.PNAS ?
April 14, 2009 ?
vol. 106 ?
no. 15 ?
Materials and Methods
Patients. Resting CD4?T cells were isolated from 6 HIV-1-infected adults who
had been continuously receiving HAART for at least 6 months and had
suppression of viremia to ?50 copies/mL.
(20). A human splenocyte cDNA library was divided into pools of 100 cDNAs
per pool. HEK293T cells were plated at 9 ? 104cells per well in 24-well dishes,
and transfected 24 h later with a total of 356 ng of DNA, including 2 ng of
phosphate method. The reference control transfection contained 350 ng
pcDNA3.1(?) (Invitrogen). At 40–48 h after transfection, the cells were lysed
in 100 ?L of passive lysis buffer (Promega) at room temperature, and centri-
fuged at 13,000 ? g at room temperature for 5 min to pellet debris. We used
20 and 10 ?L of lysate, respectively, to assay luciferase and ?-Gal activities, by
using the luciferase assay system (Promega), a chemiluminescent ?-Gal re-
porter gene assay (Roche), and luminometer (Central LB 960; Berthold) in
accordance with the manufacturers’ instructions. Fold stimulation was calcu-
lated for each sample by dividing the luciferase activity, normalized to the
?-Gal activity, by that observed in the empty vector control sample. Positive
pools were considered NF-?B-independent if their activity with the Ig?2-IFN-
GL4 reporter was ?30% of that observed with the m?B-LTR-Luc reporter, or
if they stimulated the Ig?2-IFN-GL4 reporter ?1.5-fold.
Transient Transactivation Assay in HEK293T Cells and Jurkat T Cells. In Figs. 2–4,
we used TK-RLuc (pGL4.74; Promega) as an internal control instead of pCSK-
lacZ; 20 ?L of lysates were analyzed by using the Dual-luciferase Reporter
Assay System (Promega) according to the manufacturer’s instructions. Fold
stimulation was calculated by comparing observed activities with that
achieved with the vector pmax-Empty. Jurkat T cells were grown in RPMI
medium 1640 with GlutaMax-I (Invitrogen) supplemented with 10% FBS
(Gemini Bio) and 100 U/mL each of penicillin and streptomycin. On the day of
transfection, 5 ? 105cells were plated in 2 mL in each well of a 6-well plate
?g of DNA and 9 ?L of Fugene 6. Transfections included 100 ng m?B-LTR-Luc,
50 ng TK-RLuc, and up to 2.8 ?g pmax-?VII-Ets-1; 40–48 h after transfection,
cells were lysed in 150 ?L of passive lysis buffer (Promega) for 15 min at room
Cell Lysates, Western Blots, and Antibodies. Primary CD4?T cells or HEK293T
cells were lysed in RIPA buffer (50 mM Tris, pH 7.5/150 mM NaCl/10 mM
removed by centrifugation for 20 min at 13,000 ? g in a microcentrifuge at
4 °C. Anti-Ets-1 (sc-350; Santa Cruz), anti-?-actin (Sigma), and anti-FLAG-HRP
(Sigma) were used at dilutions of 1:400, 1:2,500, and 1:3,000, respectively.
RNA Isolation and Real-Time RT-PCR. Total cellular RNA was isolated by using
RNeasy Mini Kit (Qiagen) and RT reactions were performed by using Super-
Script III Reverse Transcriptase (Invitrogen) with random primers (Invitrogen).
Expression of IFN-? or IL-2 transcripts was measured by using TaqMan Gene
mRNA was measured by RT-PCR with SYBR Green PCR Master Mix (Applied
RT were negative.
T Cell Purification and Transfections, RNA Isolation, and Real-Time RT-PCR for
were resuspended in 100 ?L of Nucleofector solution, transfected by using
program U-014, then cultured in 2 mL of RPMI medium 1640 with GlutaMax-I
?VII-Ets-1, pmax-fl-Ets-1, or pcDNA-Tat-86 was transfected. GFP expression
served as a negative control and an indicator of transfection efficiency based
on the frequency of GFP-positive cells assayed by flow cytometry. To measure
the copy number of released virus, supernatants of transfected resting CD4?
T cells were collected 72 h after nucleofection. RNA isolation and real-time
RT-PCR were done by using Amplicor Ultrasensitive for HIV-1 Kit (Roche)
following the manufacturer’s instructions.
Further details regarding the construction of reporter constructs and ex-
pression vectors are available in supporting information (SI) Materials and
ACKNOWLEDGMENTS. We thank H. Zhang for help in cell sorting, the labo-
ratory of Dr. Thomas C. Quinn for measuring viral load, and Drs. J. Blankson
by National Institutes of Health Grant AI043222 (to R.F.S.). J.L.P is a Rita Allen
Foundation Scholar and a recipient of a Kimmel Scholar Award from the
Sidney Kimmel Foundation for Cancer Research.
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