JOURNAL OF VIROLOGY, June 2007, p. 6043–6056
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 11
Sustained Induction of NF-?B Is Required for Efficient Expression of
Latent Human Immunodeficiency Virus Type 1?
Samuel A. Williams,1,2Hakju Kwon,1† Lin-Feng Chen,1‡ and Warner C. Greene1,3,4*
Gladstone Institute of Virology and Immunology1and Departments of Physiology,2Medicine,3and Microbiology and Immunology,4
University of California, San Francisco, San Francisco, California 94158
Received 18 September 2006/Accepted 12 March 2007
Cells harboring infectious, but transcriptionally latent, human immunodeficiency virus type 1 (HIV-1)
proviruses currently pose an insurmountable barrier to viral eradication in infected patients. To better
understand the molecular basis for HIV-1 latency, we used the J-Lat model of postintegration HIV-1 latency
to assess the kinetic relationship between the induction of NF-?B and the activation of latent HIV-1 gene
expression. Chromatin immunoprecipitation analyses revealed an oscillating pattern of RelA recruitment to
the HIV-1 long terminal repeat (LTR) during continuous tumor necrosis factor alpha (TNF-?) stimulation.
RNA polymerase II (Pol II) recruitment to the HIV-1 LTR closely mirrored RelA binding. Transient stimu-
lation of cells with TNF-? for 15 min induced only a single round of RelA and RNA Pol II binding and failed
to induce robust expression of latent HIV-1. Efficient formation of elongated HIV-1 transcripts required
sustained induction by NF-?B, which promoted de novo synthesis of Tat. Cyclin-dependent kinase 9 (CDK9)
and serine-2-phosphorylated RNA Pol II were rapidly recruited to the HIV-1 LTR after NF-?B induction;
however, these elongating polymerase complexes were progressively dephosphorylated in the absence of Tat.
Okadaic acid promoted sustained serine-2 phosphorylation of the C-terminal domain of RNA Pol II and
stimulated efficient transcriptional elongation and HIV-1 expression in the absence of Tat. These findings
underscore important differences between NF-?B and Tat stimulation of RNA Pol II elongation. While NF-?B
binding to the HIV-1 LTR induces serial waves of efficient RNA Pol II initiation, elongation is impaired by the
action of an okadaic acid-sensitive phosphatase that dephosphorylates the C-terminal domain of RNA Pol II.
Conversely, the action of this phosphatase is overcome in the presence of Tat, promoting very efficient RNA Pol
Therapeutic efforts to eradicate human immunodeficiency
virus type 1 (HIV-1) from infected patients with highly effec-
tive combinations of antiviral drugs have been thwarted, in
part, by a highly durable and drug-insensitive pool of latently
infected memory CD4?T cells. Latent HIV-1 infection is
characterized by integration of the provirus into the host DNA,
followed by temporary silencing of viral gene expression. Rep-
lication of these latent proviruses can be activated by various
cues, including specific antigens and select cytokines (see ref-
erence 3 for review). As a consequence of these properties,
latently infected cells are unaffected by existing antiretroviral
therapies and retain the potential to reseed systemic infection
upon activation. The observed half-life of the latent pool of
HIV-1 is at least 45 months, and assuming an initial pool of 105
infected cells, eradicating these cells would take over 60 years
of continuous treatment (13, 34). If curative therapies for
HIV-1 infection are ever to become a reality, new strategies for
rapidly eliminating or permanently silencing this pool of per-
sistently infected and drug-insensitive virus must be developed
(32). The identification of these strategies will be facilitated by
a deeper understanding of the transcriptional events underly-
ing HIV-1 latency.
The principal feature of HIV-1 latency is a failure of viral
gene expression, chiefly as a consequence of uninitiated or
aborted transcription. Expression of the integrated provirus is
regulated by the promoter and enhancer elements in the 5?
long terminal repeat (LTR) (see reference 33 for review).
Elongation of HIV-1 mRNA transcripts is strongly dependent
on the viral transactivating protein Tat, which binds to a highly
structured region of HIV-1 RNA, the trans-acting response
element, located at the 5? end of all initiated transcripts (22).
Tat recruits the cellular P-TEFb kinase complex, composed of
cyclin T1 and cyclin-dependent kinase 9 (CDK9) (44), to the
HIV-1 promoter, which in turn induces serine-2 (S2) phosphor-
ylation of proximal RNA polymerase II (Pol II) complexes
(30). This modification promotes efficient transcriptional elon-
gation. Mutations within Tat or the trans-acting response ele-
ment are responsible for latent proviruses found in U1 and
ACH-2 cell lines, respectively (10, 11). Remarkably, infectious
virus can be induced from both of these cell lines by a range of
NF-?B-inducing agents, indicating that this cellular complex
can mimic many of the effects of Tat, albeit with lower effi-
The prototypical NF-?B heterodimer composed of RelA
and p50 binds to two ?B enhancer sites in the HIV-1 LTR
located immediately upstream of the transcriptional initiation
* Corresponding author. Mailing address: Gladstone Institute of
Virology and Immunology, 1650 Owens Street, San Francisco, CA
94158. Phone: (415) 734-2000. Fax: (415) 355-0153. E-mail: wgreene
† Present address: PDL BioPharma, Inc., 34801 Campus Drive, Fre-
mont, CA 94555.
‡ Present address: Department of Biochemistry, University of Illi-
nois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL
?Published ahead of print on 21 March 2007.
site (4, 31, 41). Induction of NF-?B is associated with both
enhanced transcriptional initiation and elongation of HIV-1
mRNA transcripts (28, 39). RelA contains two strong tran-
scriptional activation domains which mediate recruitment of a
number of positively acting transcription factors, including P-
TEFb (2). RelA-driven recruitment of P-TEFb is thought to
underlie initial elongation of HIV-1 mRNA transcripts, per-
mitting synthesis of Tat (2). Our recent studies further dem-
onstrate that RelA contributes to transcriptional initiation by
displacing repressive p50-HDAC1 complexes bound to ?B
sites of the HIV-1 LTR under basal conditions (41).
In the absence of activating stimuli, NF-?B is predominantly
localized within the cytoplasmic compartment and prevented
from binding DNA by association with inhibitory I?B proteins
(8, 14, 18, 42). Stimulus-coupled activation of NF-?B involves
increased activity of the IKK signalsome complex, which pro-
motes phosphorylation of I?B?, a signal for polyubiquitylation
and proteasome-mediated degradation of the inhibitor (9, 27,
35, 43). Liberated NF-?B complexes rapidly translocate into
the nucleus, bind cognate DNA enhancers, and induce gene
expression. Among NF-?B’s many cellular targets is the I?B?
gene. Thus, NF-?B activation stimulates de novo synthesis of
its own inhibitor, leading to auto-regulation of NF-?B activity
(35). In the presence of continuous stimulation of cells, NF-?B
induction occurs in a dampened oscillatory manner involving
steadily decreasing waves of NF-?B moving into the nucleus (7,
These features of NF-?B activation and the apparent func-
tional homology of RelA and Tat as recruiters of P-TEFb
prompted us to further investigate the role of NF-?B in the
activation of latent HIV-1 gene expression. Owing to the rarity
of latently infected cells in HIV-1-infected patients and the
absence of specific surface markers for purification of these
cells, we employed the J-Lat model of HIV-1 latency (21).
J-Lats are Jurkat-derived T-cell clones infected with a single
copy of a full-length HIV-1 provirus engineered to express
green fluorescent protein (GFP) in lieu of Nef. J-Lat cells
produce essentially no GFP under basal conditions, reflecting
an absence of detectable RNA Pol II binding to the latent
promoter. However, the transcriptional quiescence of HIV-1
in these cells can be reversed by treatment with tumor necrosis
factor alpha (TNF-?), which promotes RNA Pol II binding
(41). These findings implicate a deficiency in transcriptional
initiation as a primary feature of the HIV-1 latency observed in
many J-Lat clones.
In the current study, we compared and contrasted changes in
RNA Pol II elongation in latently infected cells when NF-?B-
inducing stimuli were administered transiently or continuously
and when Tat expression was permitted or inhibited.
MATERIALS AND METHODS
Cell lines and culture conditions. J-Lat 6.3 cells were maintained in RPMI
supplemented with 10% fetal calf serum, penicillin, streptomycin, and L-glu-
tamine. For stimulation, cells were treated with 20 ng/ml TNF-? (R&D Systems)
or 30 nM okadaic acid (OA; Biomol) alone or in combination. For inhibition
experiments, 5,6-dichloro-1-?-D-ribofuranosylbenzimidazole (DRB; Sigma) was
used at 10 ng/ml, and cycloheximide (Sigma) was used at 10 ?g/ml. To produce
NF-?B–DsRed2 reporter cells, Jurkat or various J-Lat cells were transduced with
a lentiviral vector, PPT-?B-DsRed2 (12). Briefly, this construct was cotransfected
with the vesicular stomatitis virus G protein envelope into HEK 293T cells, and
virus-containing supernatants were harvested and concentrated with Centricon
filtration units (Millipore). Parental Jurkat or J-Lat 6.3, 8.4, or 15.2 cells were
infected with concentrated virus stocks incubated for 72 h, and DsRed2-negative
cells were sorted by fluorescence-activated cell sorting. After negative sorting,
cells were treated with TNF-? and incubated for 24 h, and cells with TNF-?-
inducible DsRed2 expression were selected by flow cytometry and designated
J?Red, J6.3?Red, J8.4?Red, and J15.2?Red.
Chromatin immunoprecipitation. J-Lat 6.3 cells were adjusted to 1 ? 106
cells/ml and incubated in medium with or without TNF-? (20 ng/ml) for various
times. For pulse-stimulation experiments, cells were treated with TNF-? for 15
min, washed three times in complete culture medium, and then returned to
culture. Chromatin immunoprecipitation (ChIP) assays were performed as de-
scribed elsewhere (40). Antibodies employed were RelA (sc-109), RNA Pol II
(sc-899), and CDK9 (H-169) (all three from Santa Cruz Biotechnology) and
ser-2-RNA polymerase II (H5) and ser-5-RNA Pol II (H19) (Covance).
To detect specific HIV-1 LTR and ?-actin unique short DNA regions, PCR
primers were as described in reference 41. HIV?1000 5?HIV1000 (5?-TATTG
CACCAGGCCAGATGA-3?) and 3?HIV1200 (5?-GTCTCTAAAGGGTTCCT
TTG-3?). HIV?3000 was amplified with 5?HIV3000 (5?-ACTGGCAGAAAAC
HIV?5000 DNA was amplified with 5?HIV5000 (5?-CATCATGGATCCACTC
GACAGAGGAGAG-3?) and HIV3?5200 (5?-ATGATGAAGCTTGAGTCTG
ACTGTTCTG-3?). HIV?7000 was amplified with 5?HIV7000 (5?-AGTGACA
CAATCACCCTCCC-3?) and 3?HIV7200 (5?-ATAATTCACTTCTCCAATTG-
3?). Amplification was performed with Taq polymerase (QIAGEN) for 35 to 40
cycles, and products were analyzed on 2.5% agarose gels. Images were acquired
using an EagleEye II digital imaging system (Stratagene). All ChIP extracts were
assessed for enrichment in ?-tubulin DNA as a control for nonspecific DNA
binding. Specific enrichment in target DNA sequences by a specified antibody
was interpreted as DNA binding by the antibody target. ChIP extracts were
quantitated with the QuantiTect SYBR Green PCR kit (QIAGEN). Fluores-
cence profiles were collected on an ABI 7700 real-time thermal cycler and
analyzed with SDS v1.91 (Applied Biosystems).
RNA extraction and analysis of initiated and elongated HIV-1 transcripts.
J-Lat 6.3 cells (1 ? 106cells/ml) were either not treated or pretreated with OA
(100 nM) for 1 h at 37°C and were either not stimulated or stimulated with
TNF-? (20 ng/ml) for 30 min. RNA was extracted from 5 ? 106cells via
RNAWiz (Ambion), and transcripts were quantitated using the QuantiTect
SYBR Green reverse transcription-PCR (RT-PCR) kit (QIAGEN). To quan-
titate viral transcripts, serial dilutions of a quantitated RNA stock of full-
length HIV-1 genome were employed as a reference standard (gift from R.
Grant, Gladstone Institute of Virology and Immunology). PCR primers for
detection of initiated and elongated HIV-1 and ?-actin transcripts were as
described previously (41). Fluorescence profiles were collected on an ABI
7700 real-time thermocycler and analyzed with SDS v1.91 (Applied Biosys-
tems). The absence of nonspecific bands in RT-PCR products was confirmed
by electrophoresis of samples on 2% agarose gels and visualization with
ethidium bromide and UV light.
Immunoblotting analysis. For analysis of protein expression, J-Lat 6.3 cells
were collected by centrifugation and lysed on ice in egg lysis buffer (50 mM
HEPES, pH 7, 250 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) for 20 min, and
lysates were clarified by microcentrifugation. Nuclear and cytoplasmic extracts
were prepared as described previously (1). The protein concentration was quan-
titated using the Bradford protein assay (Bio-Rad), and 10 ?g of each sample was
added to an equal volume of 2? Laemmli buffer and heated to 95°C for 5 min.
Samples were separated on 10% acrylamide Tris-HCl-buffered sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gels (Bio-Rad), transferred to poly-
vinylidene difluoride membranes, and immunoblotted with antibodies specific
for Sp1, RelA, or I?B? (Santa Cruz) or ?-tubulin or HIV-1 Tat (Advanced
Bioscience Laboratories). For detection of HIV-1 Tat, samples were concen-
trated by immunoprecipitation with antibodies recognizing HIV-1 Tat (116P;
Covance) before immunoblotting.
Transfection, nucleofection, and flow cytometric detection of transfected cells.
For DNA transfection by electroporation, 1 ? 107J-Lat 6.3 cells were resus-
pended in 400 ?l of culture medium with 5 ?g of FLAG-Tat, T7-RelA, or empty
pCMV4 expression vector and 1 ?g pMACS H-2Kkmarker DNA and transferred
to a 0.4-cm gap electroporation cuvette (Bio-Rad). Cells were electroporated
with a Bio-Rad Gene Pulser II set to 0.975 ?F and 250 V and returned to culture
for 16 h before further manipulation. To identify transfected cells, cells were
stained with biotin–anti-H-2Kkantibodies, washed, and counterstained with
streptavidin-allophyocyanin (Pharmingen). For nucleofection, J-Lat cells were
resuspended at a concentration of 5 ? 107cells/ml in 100 ?l of reagent R
(Amaxa) with 5 ?g of high-performance liquid chromatography-purified, an-
nealed RNA oligonucleotides siTat S (5?-CUGCUUGUACCAAUUGCUAdT
6044WILLIAMS ET AL.J. VIROL.
dT-3?), AS (5?-UAGCAAUUGGUACAAGCAGdTdT-3?), siTatMM S (5?-
CUGCUUGUCACAAUUGCUAdTdT-3v), and AS (5?-UAGCAAUUGUGA
CAAGCAGdTdT-3?) (Ambion). Nucleofections were performed using program
O28 with an Amaxa nucleofector. Small interfering RNA (siRNA)-nucleofected
cells did not require marking, as 95% of cells were routinely nucleofected with
siRNA. Cells were analyzed with a FACSCalibur flow cytometer (Becton Dick-
inson) and FlowJo software (Treesoft).
Continuous stimulation of NF-?B drives oscillating but syn-
chronous association of RelA and RNA polymerase II with the
latent HIV-1 LTR. Our prior studies of the latent HIV-1 LTR
revealed an absence of RNA polymerase II binding under
unstimulated conditions but effective recruitment following
TNF-? activation of the J-Lat cells. To further characterize the
events underlying activation of the latent HIV-1 LTR, we
sought to compare the kinetics of RelA and RNA Pol II re-
cruitment after TNF-? stimulation of these cells. ChIP assays
were employed to study the interaction of these factors with
the HIV-1 LTR in J-Lat cells in vivo. Sheared formaldehyde-
cross-linked chromatin extracts prepared from J-Lat cells un-
treated or treated with 20 ng/ml TNF-? continuously for up to
6 h were immunoprecipitated with antibodies specific for ei-
ther RelA or RNA Pol II. The abundance of HIV-1 LTR DNA
in immunoprecipitates was assessed by ethidium bromide vi-
sualization of PCR products amplified with primers specific for
the HIV-1 LTR. Consistent with prior results (41), neither Rel
A nor RNA Pol II binding to HIV-1 LTR DNA was detected
in untreated cells (Fig. 1A). However, both RelA and RNA Pol
II binding were detected 15 min after stimulation with TNF-?,
and continued binding was observed after 30 min. ChIP anal-
yses performed after 1 h of stimulation revealed a synchronous
diminution of RNA Pol II and RelA binding. In the continuous
presence of TNF-?, a second wave of RelA and RNA Pol II
binding was observed at 2 h, followed by a less pronounced
decline at 6 h. Importantly, levels of input HIV-1 LTR DNA
were similar at each time point, and analysis of nonspecific
DNA sequences supported specificity in the ChIP procedure
(Fig. 1A and data not shown).
The dynamic pattern of RelA association with the latent
HIV-1 LTR after TNF-? stimulation resembled oscillatory
patterns of NF-?B recruitment recently described for various
NF-?B target genes (7, 29, 38). This oscillation is attributed to
NF-?B-dependent induction of I?B? gene expression, which
inhibits DNA binding and promotes nuclear export of RelA.
Continued stimulation of the upstream I?B kinases induces
secondary rounds of I?B? phosphorylation and degradation,
leading to delayed waves of nuclear accumulation of NF-?B.
To examine whether this process might underlie the pattern of
RelA recruitment to the latent HIV-1 LTR, the abundance of
I?B? and nuclear RelA after TNF-? stimulation was assessed
by immunoblot analysis. After TNF-? stimulation, I?B? abun-
dance was rapidly ablated; however, resynthesis of the inhibi-
tory protein was apparent after 1 h. The level of I?B? expres-
sion after 2 h of TNF-? treatment was slightly depressed
relative to untreated samples (Fig. 1B). Nuclear abundance of
RelA was inversely correlated to I?B? levels, with a peak in
nuclear RelA occurring after 15 min of TNF-? stimulation, a
nadir at 1 h, and a second peak at 2 h of stimulation (Fig. 1C).
This kinetic pattern of nuclear RelA expression was strikingly
similar to the ChIP analyses of RelA and RNA Pol II binding
to HIV-1 LTR DNA, suggesting that the oscillating nuclear
abundance of RelA likely underlies the temporally dependent
recruitment of Rel A and RNA Pol II observed in ChIP anal-
Transient activation of NF-?B induces a single round of
RelA and RNA polymerase II binding to the latent HIV-1 LTR.
We next investigated whether a transient pulse of TNF-? could
be employed to induce a single cycle of RelA activity. To test
this possibility, J-Lat 6.3 cells were stimulated with TNF-? for
FIG. 1. TNF-? stimulation induces an oscillating pattern of RelA
and RNA Pol II binding to the latent HIV-1 LTR. (A) TNF-? stim-
ulation leads to synchronous but oscillating recruitment of RelA and
RNA Pol II to the latent HIV-1 LTR. Fixed chromatin extracts from
J-Lat 6.3 cells either untreated or treated with 20 ng/ml TNF-? for
various times were immunoprecipitated with antibodies specific for
RelA, RNA Pol II, or no antibody as a control. Immunoprecipitates
were assessed for enrichment in HIV-1 LTR DNA by UV visualization
of PCR products in an ethidium bromide-stained gel. Data are repre-
sentative of three independent experiments. Note the synchronicity of
RelA and RNA Pol II recruitment to the HIV-1 LTR, as well as the
nadir in binding 1 h after TNF-? stimulation. Quantitation of enrich-
ment (fold increase) above the “no-antibody” control is indicated
below each sample. (B) TNF-?-induced recruitment of RelA and RNA
Pol II to the latent HIV-1 LTR coincides with I?B? degradation.
Cytoplasmic extracts of samples treated as for panel A were prepared,
and I?B? levels were assessed by immunoblotting. Cytoplasmic levels
of ?-tubulin were assessed to confirm equivalent loading of samples.
(C) TNF-? induces bimodal nuclear enrichment of RelA. Nuclear
extracts of samples treated as for panel A were prepared and analyzed
for RelA enrichment by immunoblotting. Nuclear Sp1 levels were
assessed to confirm equivalent loading. Note the largely overlapping
patterns of nuclear enrichment of RelA and its recruitment to the
latent HIV-1 LTR as assessed by ChIP (A).
VOL. 81, 2007 EXPRESSION OF HIV-1 REQUIRES SUSTAINED NF-?B6045
15 min, washed extensively, and returned to culture for various
times. Immunoblot analysis of cytoplasmic I?B? expression
demonstrated that the pulsed administration of TNF-? in-
duced rapid degradation of the NF-?B inhibitor, with overall
levels falling to levels similar to those observed with continuous
TNF-? stimulation (Fig. 2A). Resynthesis of I?B? was appar-
ent 1 h after TNF-? pulse treatment, and in contrast to con-
tinuous stimulation, expression levels thereafter appeared to
remain slightly higher than in lysates of untreated cells. Cor-
respondingly, early nuclear accumulation of RelA was ob-
served in TNF-?-pulsed samples with kinetics similar to those
in samples treated continuously with TNF-? (Fig. 2B). Signif-
icantly, TNF-? pulse-stimulation did not induce a second wave
of nuclear RelA after 2 h of chase. Taken together, these
results indicate that delayed waves of nuclear RelA are not
induced by transient TNF-? stimulation.
To examine the effects of transient induction of NF-?B on
transcriptional regulation of latent HIV-1, ChIP analyses of
RelA and RNA Pol II binding to HIV-1 LTR DNA were
conducted in TNF-?-pulsed samples. Consistent with the pre-
dicted transient induction of nuclear NF-?B expression,
TNF-? pulse treatment induced a single round of RelA re-
cruitment to HIV-1 LTR DNA, with a peak in binding occur-
ring at 15 min (Fig. 2C). Pulse-stimulation with TNF-? also
induced only a single round of RNA Pol II binding to the
HIV-1 LTR DNA, which appeared to peak at 30 min. Our
earlier studies showed that either RelA recruitment or TSA
stimulation results in increased acetylation of histones sur-
rounding the HIV-1 LTR (41). Thus, changes in chromatin
structure likely contribute to the increased binding of RNA
Transient induction of NF-?B fails to activate latent HIV-1
proviral gene expression. The transient nature of RNA Pol II
recruitment to HIV-1 LTR DNA after TNF-? pulse-stimula-
tion suggested that these stimulation conditions might not in-
duce exit of HIV-1 proviruses from latency in J-Lat cells. To
test this possibility, J-Lat 6.3 cells were either transiently or
continuously stimulated with TNF-? and cultured for 16 h, and
HIV-1-dependent expression of the viral GFP reporter (in-
serted in lieu of Nef) was assessed by flow cytometry. Expres-
sion of GFP in unstimulated J-Lat cells was observed in less
than 0.5% of cells (Fig. 3A). Continuous stimulation with
TNF-? induced robust GFP expression in 36% of the J-Lat
cells. In contrast, TNF-? pulse-stimulation induced GFP ex-
pression in only 3.8% of cells. Similar results were observed in
J-Lat clones 8.4 and 15.2, indicating that this effect can be
generalized beyond a single J-Lat clone (data not shown). To
test the effectiveness of TNF-? pulse-stimulation in the activation
of a ?B-dependent integrated gene, induction of DsRed2 expres-
sion was assessed in an independent set of Jurkat cells stably
transduced with an integrated 4x? B-DsRed2 reporter vector
but lacking HIV-GFP proviruses (J?Red cells). In the absence
of stimulation, DsRed2 expression in J?Red cells was largely
suppressed, although a low level of constitutive reporter activ-
ity was detected (Fig. 3B). After 16 h of continuous TNF-?
stimulation, 98% of the J?Red cells expressed DsRed2, indi-
cating a nearly uniform induction of ?B-specific gene expres-
sion in these cells. Similarly, and in striking contrast to expres-
sion of HIV-1-driven GFP, 95% of J?Red cells pulsed for 15
min with TNF-? exhibited DsRed2 expression 16 h after in-
To exclude the possibility of confounding mutations and to
confirm that similar transient inducibility of ?B-dependent
gene expression is possible within J-Lat cells, populations of
J-Lat 6.3 cells were stably transduced with the ?B-DsRed2
reporter and then pulsed or continuously stimulated with
TNF-? and analyzed by flow cytometry after 16 h of culture. As
observed in J?Red cells, J6.3?Red cells exhibited a modest
background of DsRed2-expressing cells (Fig. 3C). In cells con-
tinuously stimulated with TNF-?, 97% expressed ?B-driven
DsRed2, and 30% expressed HIV-1 LTR-driven GFP. Simi-
larly, 93% of TNF-?-pulsed J6.3?Red cells exhibited DsRed2
FIG. 2. Transient induction of NF-?B induces unimodal recruit-
ment of RNA polymerase II to the latent HIV-1 LTR. (A) Pulsed
administration of TNF-? induces I?B? degradation. J-Lat 6.3 cells
were stimulated with 20 ng/ml TNF-? or left untreated for 15 min,
washed twice in medium, and returned to culture for various times.
Cytoplasmic extracts were prepared, and I?B? levels were assessed by
immunoblotting. Cytoplasmic ?-tubulin levels were assessed to con-
firm equivalent loading of samples. Note the similarity in depletion of
I?B? in transiently and continuously TNF-? treated samples (Fig. 1B).
(B) Pulsed administration of TNF-? induces transient activation of
NF-?B. Nuclear extracts of samples treated as for panel A were pre-
pared and analyzed for recruitment of RelA by immunoblotting. Nu-
clear Sp1 levels were assessed to confirm equivalent loading.
(C) Pulsed TNF-? administration induces a unimodal pattern of RelA
and RNA Pol II recruitment to the latent HIV-1 LTR. Fixed chroma-
tin extracts of samples treated as for panel A were prepared and
subjected to immunoprecipitation with antibodies specific to RelA or
RNA Pol II or without antibody, as a nonspecific control. Immuno-
precipitates were assessed for enrichment in HIV-1 LTR DNA by UV
visualization of PCR products in a gel stained with ethidium bromide.
Data are representative of three separate experiments. Note the ab-
sence of a second wave of RNA Pol II recruitment to the latent HIV-1
LTR. Quantitation of enrichment (fold increase) above the no-anti-
body control is indicated below each sample.
6046 WILLIAMS ET AL.J. VIROL.
expression, but only 1.7% of these cells expressed HIV-1 LTR-
GFP. Similar results were obtained in J8.4?Red and
J15.2?Red cells. Thus, failure of robust expression of HIV-1 in
response to transient NF-?B induction is not a general feature
of ?B-dependent transcription but rather appears to reflect
properties unique to the latent HIV-1 LTR.
TNF-? induction of efficient RNA Pol II elongation on the
HIV-1 LTR requires prolonged stimulation. To further explore
differences at the RNA level occurring in J-Lat cells after
transient or continuous stimulation with TNF-?, the levels of
initiated versus elongated HIV-1 mRNA transcripts were as-
sessed by quantitative real-time RT-PCR. Continuous TNF-?
FIG. 3. Transient induction of NF-?B is sufficient to induce robust general ?B-dependent, but not latent HIV-1, gene expression. (A) Transient
TNF-? administration induces poor expression of latent HIV-1. J-Lat 6.3 cells were left untreated or stimulated with 20 ng/ml TNF-? continuously
or for 15 min, followed by washing and continued culture. HIV-LTR-dependent expression of GFP was assessed by flow cytometry. Note the
overall lack of GFP expression in samples transiently treated with TNF-?. (B) Transient induction of NF-?B is sufficient to stimulate general
?B-dependent gene expression. J?Red cells were treated as for panel A, and ?B-dependent expression of DsRed2 was assessed by flow cytometry.
Note the strong induction of ?B-dependent gene expression by transient TNF-? stimulation. (C) Transient NF-?B induction induces robust
expression of ?B-dependent genes, but not latent HIV-1, in J-Lat 6.3 cells. J6.3?Red cells were treated as for panel A, and HIV-1 LTR-dependent
expression of GFP and ?B-dependent expression of DsRed2 were assessed by flow cytometry. Note the strong induction of ?B-dependent
gene expression and relative absence of HIV-1 gene expression induced by transient TNF-? stimulation.
VOL. 81, 2007 EXPRESSION OF HIV-1 REQUIRES SUSTAINED NF-?B 6047
treatment induced a rapid 10-fold increase in initiated HIV-1
mRNA transcripts. These plateaued temporarily at 1 h and
then increased (Fig. 4A). Conversely, elongated HIV-1 mRNA
transcripts were only modestly induced during the first 4 h of
TNF-? treatment, with exponential increases occurring there-
after. Extrapolation of the rate of transcript accumulation re-
vealed a similar rate of initiated HIV-1 transcript accumulation
in early and late transcription (Fig. 4C). In contrast, elongated
HIV-1 transcripts fail to accumulate at an appreciable rate
until after 4 h of TNF-? stimulation.
Transient activation of NF-?B with TNF-? pulse-stimulation
induced initiated HIV-1 mRNA transcripts with kinetics sim-
ilar to those observed at early time points in continuously
stimulated cells; however, after 1 h, initiated transcript abun-
dance declined rapidly, with a t1/2of 60 min (Fig. 4B). Elon-
gated HIV-1 mRNA transcripts in TNF-?-pulsed samples were
modestly induced in the first hour; however, sustained induc-
tion was not observed, and after 2 h the abundance of elon-
gated transcripts declined to less than one copy per cell. These
findings indicate that transient induction of NF-?B is sufficient
to elicit a brief but temporary increase in transcriptional activ-
ity of the latent HIV-1 provirus. Continuous activation of the
cells with TNF-? sharply increases the number of initiated
HIV-1 transcripts; however, marked increases in HIV-1 elon-
gation do not occur until after 4 h of stimulation.
TNF-? induction of HIV-1 Tat synergizes with active NF-?B
to promote efficient transcriptional elongation of latent HIV-1
mRNA. The delayed accumulation of elongated HIV-1 mRNA
transcripts relative to initiated transcripts in TNF-?-stimulated
J-Lat cells suggested that efficient elongation is dependent on
de novo synthesis of an NF-?B-responsive factor. Alterna-
tively, binding of RelA to the HIV-1 LTR might slowly induce
chromatin rearrangements required for effective elongation.
To distinguish between these possibilities, new protein syn-
thesis in J-Lat 6.3 cells was inhibited with the translational
inhibitor cycloheximide, and initiated and elongated HIV-1
transcripts were quantitated after TNF-? stimulation. Cyclo-
heximide strongly impaired resynthesis of I?B? in TNF-?-
treated cells, confirming its bioactivity in these assays (data not
shown). Early initiated and elongated HIV-1 transcript levels
were modestly enhanced in cycloheximide-treated J-Lat cells
stimulated with TNF-? continuously for 1 h, indicating that
these early transcripts are not dependent on de novo protein
synthesis (Fig. 5A). In contrast, accumulation of elongated
HIV-1 transcripts in samples treated with TNF-? for 6 h was
markedly reduced in the presence of cycloheximide. These
findings indicate that the initial NF-?B induction of transcrip-
tional initiation and low-level elongation of HIV-1 mRNA do
not require de novo protein synthesis while the high-efficiency
elongation occurring after 4 to 6 h is dependent on new protein
synthesis in J-Lat cells. The increased levels of transcriptional
initiation induced in cycloheximide-treated cells are likely a
consequence of the inhibition of I?B? resynthesis, resulting in
prolonged NF-?B activity. Levels of ?-actin mRNA and cell
viability remained stable for the duration of the experiment,
suggesting cycloheximide-induced toxicity was not a factor in
the observed impairment of elongated HIV-1 mRNA tran-
script accumulation (data not shown).
The restricted transcriptional elongation apparent at early
time points after TNF-? stimulation could reflect limited ex-
pression of Tat, which is under control of the viral LTR and
regulated by NF-?B. Immunoblot analysis of Tat expression in
J-Lat cells stimulated with TNF-? did not detect Tat at 0, 0.25,
0.5, and 1 h after induction. Low levels of Tat were first de-
tected at 2 h, and higher levels were found at 4, 6, and 18 h
(Fig. 5B). Of note, this slow accumulation of Tat tracked the
observed delay in transcriptional elongation noted in Fig. 4B,
suggesting that an absence of Tat may underlie the lack of
latent gene expression in TNF-?-pulse-treated cells.
To examine whether expression of Tat alone was sufficient to
rescue expression of latent HIV-1 in response to transient
NF-?B induction, J-Lat 6.3 cells were transfected with control,
Tat, or RelA expression vectors, pulse-stimulated or continu-
ously stimulated with TNF-?, and analyzed by flow cytometry
for HIV-directed GFP expression. Ectopic expression of Tat
induced HIV-1 LTR-driven GFP expression in 9.8% of cells in
the absence of stimulation (Fig. 5C). In contrast, ?70% of
Tat-transfected cells either pulse-stimulated or continuously
stimulated with TNF-? expressed GFP. Of note, mean GFP
FIG. 4. Efficient elongation of HIV-1 mRNA transcripts is delayed
in TNF-?-activated J-Lat cells. (A) TNF-? treatment of J-Lat cells
induces rapid accumulation of initiated, but not elongated, HIV-1
mRNA transcripts. J-Lat 6.3 cells were treated with 20 ng/ml TNF-?
for various times, and total RNA was extracted. Initiated and elon-
gated HIV-1 mRNA transcripts were quantitated by real-time RT-
PCR. Note the delayed emergence of elongated HIV-1 transcripts
relative to the rapid increase in initiated transcripts. (B) Transient
induction of NF-?B does not induce accumulation of elongated HIV-1
mRNA transcripts. J-Lat 6.3 cells were treated with 20 ng/ml TNF-?
for 15 min, washed twice, and returned to culture for various times.
Initiated and elongated HIV-1 mRNA transcript abundance were as-
sessed as for panel A. (C) The kinetics of initiated and elongated
HIV-1 transcript formation in TNF-?-induced J-Lat cells are dynamic.
The rate of transcript formation in continuously TNF-?-stimulated
J-Lat 6.3 cells was determined from the data in panel A. Note that the
rate of initiated HIV-1 mRNA transcript formation is relatively con-
stant across time, in contrast to the accelerating rate of elongated
6048WILLIAMS ET AL.J. VIROL.
fluorescence in J-Lat cells transfected with Tat and continu-
ously stimulated with TNF-? was threefold higher than that
observed in TNF-? pulse-stimulated cells (data not shown).
These findings indicate that the initial wave of NF-?B recruited
to the HIV-1 LTR is sufficient to drive effective gene expres-
sion in the context of Tat expression and argues against the
necessity for a second wave of NF-?B recruitment. Impor-
tantly, ectopic expression of RelA strongly drove expression of
latent HIV-1 without TNF-?, demonstrating inducibility of
transfected cells in the absence of stimulation.
To further confirm a role for Tat as the key NF-?B-induced
factor mediating TNF-?-induced HIV-1 gene expression, we
examined the effect of Tat “knockdown” with siRNAs specific
to the viral transactivator. Tat siRNA, but not a mismatched
siRNA control, decreased TNF-?-induced Tat expression to
undetectable levels (Fig. 5D). Tat siRNA reduced expression
of HIV-GFP in J-Lat cells continuously stimulated with TNF-?
from 24.7% to 3.8% but did not affect early HIV-1 transcrip-
tional initiation (Fig. 5E). Taken together with studies of Tat
overexpression, these studies demonstrate that expression of
Tat is both necessary and sufficient for efficient TNF-?-induced
expression of HIV-1 in latently infected cells.
TNF-? induces recruitment of CDK9 to the latent HIV-1
LTR. Elongation of HIV-1 mRNA is strongly dependent on
phosphorylation of the carboxy-terminal domain (CTD) of
RNA Pol II by P-TEFb (19), a factor recruited by Tat to the
HIV-1 LTR. Recent studies suggested that RelA is similarly
capable of recruiting P-TEFb to ?B-responsive genes, such as
interleukin-8 (2); however, the role of NF-?B-dependent P-
TEFb recruitment to the HIV-1 LTR has not been explored.
To determine whether early TNF-?-induced elongation of
HIV-1 mRNA transcripts is dependent on P-TEFb or is rather
FIG. 5. TNF-?-induced expression of HIV-1 is dependent on de novo synthesis of Tat. (A) TNF-?-induced accumulation of elongated, but not
initiated, HIV-1 mRNA transcripts is dependent on de novo protein synthesis. J-Lat 6.3 cells were preincubated with 10 ?g/ml cycloheximide for
30 min or left in complete culture medium before a 15-min pulse or continuous stimulation with TNF-? for 1 or 6 h. Total RNA was extracted
and initiated, and elongated HIV-1 mRNA transcripts were quantitated by real-time RT-PCR. Note the continued accumulation of initiated HIV-1
transcripts in cycloheximide-treated samples at both 1 and 6 h after TNF-? stimulation, whereas accumulation of elongated transcripts is blunted
by cycloheximide at 6, but not 1, h after TNF-? treatment. Data are representative of three independent experiments. (B) Expression of Tat is
delayed in TNF-?-stimulated J-Lat cells. Tat was immunoprecipitated from whole-cell lysates of J-Lat 6.3 cells treated with TNF-? for various
times, and expression levels were assessed by immunoblotting. Note that efficient elongation of HIV-1 transcripts in Fig. 4A coincides with the
kinetics of Tat expression. (C) Ectopic expression of HIV-1 Tat rescues HIV-1 gene expression in response to transient TNF-? stimulus. J-Lat 6.3
cells were cotransfected with control empty CMV, Tat, or RelA expression vectors and a plasmid expressing the cell surface H-2Kkmarker to
identify transfected cells. Transfected cells were stimulated with TNF-? for 15 min or continuously, and GFP expression was assessed in the
H-2Kk-expressing cells. (D) Tat is required for efficient TNF-?-induced expression of latent HIV-1. J-Lat 6.3 cells were nucleofected with siRNA
targeting Tat mRNA or a mismatched sequence, and knockdown of Tat expression was confirmed by immunoblotting (bottom panel). siRNA-
treated cells were stimulated with TNF-?, and the percentage of cells expressing HIV-GFP was quantitated by flow cytometry (top). (E) Total RNA
was extracted from cells treated as for panel D, and initiated (left) or elongated (right) transcripts were quantitated by RT-PCR.
VOL. 81, 2007EXPRESSION OF HIV-1 REQUIRES SUSTAINED NF-?B 6049
a consequence of “slip-through” elongation of unphosphory-
lated polymerase complexes, we examined the effect of the
P-TEFb inhibitor DRB on elongated HIV-1 mRNA transcript
formation in cells stimulated with TNF-? for 1 h. As a positive
control, DRB inhibition of delayed Tat-dependent elongation
of HIV-1 mRNA was assessed in samples treated with TNF-?
for 6 h. Prolonged TNF-? stimulation induced accumulation of
?500 initiated and ?80 elongated transcripts per cell (Fig. 6A
and B). Addition of DRB did not markedly alter the number of
initiated transcripts but did greatly reduce the number of elon-
gated transcripts. Samples treated with TNF-? for 1 h accu-
mulated ?90 initiated transcripts and ?4 elongated transcripts
per cell. DRB treatment had little effect on the accumulation
of initiated HIV-1 mRNA transcripts in samples treated for
1 h, whereas elongated transcript formation was reduced to
levels below the limit of detection (Fig. 6A and B). These
findings suggest that both early Tat-independent and late Tat-
HIV-1 mRNA likely depend on active P-TEFb and that P-
TEFb is recruited to the HIV-1 LTR quickly after TNF-?
stimulation before Tat is synthesized, likely by NF-?B. ChIP
analyses of CDK9 binding to the HIV-1 LTR in TNF-?-stim-
ulated J-Lat cells further supported this notion (Fig. 6B).
CDK9 binding to HIV LTR DNA was not detected in unstimu-
lated cells; however, abundant enrichment was noted within 15
min of TNF-? stimulation and followed the oscillating pattern
observed with RelA and RNA Pol II (Fig. 6C). CDK9 binding
to the HIV LTR exhibited kinetics similar to those of RelA
recruitment and certainly preceded expression of Tat (Fig.
5C). These findings are consistent with P-TEFb recruitment to
the HIV LTR by RelA.
TNF-? induces rapid association of serine-2 phosphoryla-
tion of RNA polymerase II with the HIV-1 LTR but not distal
HIV-1 DNA. The observed early recruitment of P-TEFb to the
HIV LTR was somewhat surprising in light of the restricted
elongation of HIV-1 mRNA observed at early time points.
P-TEFb phosphorylates S2 residues within the CTD of RNA
Pol II, a modification that promotes transcriptional elongation.
We hypothesized that P-TEFb directed to the HIV-1 LTR
might have reduced access to RNA Pol II relative to P-TEFb
recruited by Tat and would consequently serve as a “weaker”
kinase. S2 phosphorylation of the CTD of RNA Pol II bound
at proximal or distal sites on the HIV-1 proviral DNA in
TNF-?-treated J-Lat 6.3 cells was assessed by ChIP. However,
binding of phospho-S2 RNA Pol II to proximal sites in the
HIV-1 LTR DNA was readily detected at 15 and 30 min after
TNF-? stimulation (Fig. 6B, left panel) and was followed by a
decline at 1 h that paralleled the decline in RelA binding.
Binding increased at 2, 4, and 6 h in cells continuously stimu-
lated with TNF-?, returning to levels observed in 15- and
30-min samples. The similarity in RNA Pol II phosphorylation
at early and late time points suggests that the kinase complex
driving phosphorylation of RNA Pol II on the HIV-1 LTR has
similar activity when directed by RelA or by Tat. Conse-
quently, the inefficiency of elongation in early TNF-?-induced
HIV-1 mRNA transcription is unlikely to be solely the conse-
quence of insufficient P-TEFb kinase activity localized to the
LTR or its ability to modify the CTD of RNA Pol II.
To further explore the disparity in transcriptional outcomes
in early and late TNF-?-induced transcriptional elongation of
HIV-1 mRNA, the binding of RNA Pol II and phospho-S2
RNA Pol II to regions downstream of the HIV-1 LTR was
assessed. ChIP analysis of RNA Pol II binding to coding se-
quences of HIV-1 DNA 5,000 bp downstream (HIV?5000
DNA) of the transcription start site revealed detectable bind-
ing of the polymerase 15 and 30 min after TNF-? induction.
This binding was followed by a characteristic decrement at 1 h
(like RelA) and a second wave of binding at 2, 4, and 6 h (Fig.
6C, right panel). In contrast, binding of phospho-S2-RNA Pol
II to HIV?5000 DNA was markedly limited for the first 2 h of
TNF-? induction but became readily apparent at later time
points where effective Tat production occurred (Fig. 5B). The
combined observations of efficient phosphorylation of RNA
Pol II at the proximal HIV-1 LTR and the absence of phos-
phorylation at downstream coding regions following early
TNF-? stimulation suggest that RNA Pol II is progressively
dephosphorylated during elongation. Further, the restoration
of downstream RNA polymerase II phosphorylation during
late TNF-?-induced transcription suggests that this dephos-
phorylation is somehow overcome in the presence of Tat.
To further test this possibility and to exclude potential target
site-specific effects, a panel of HIV-1 coding regions composed
of ?1,000, ?3,000, ?5,000, and ?7,000 bp downstream of the
HIV-1 transcriptional initiation site were examined for binding
by RNA Pol II and phospho-S2-RNA Pol II. Samples stimu-
lated with TNF-? for 30 min revealed a progressive reduction
of RNA Pol II and phospho-S2-RNA Pol II at successively
distal coding regions (Fig. 6D and E). Notably, phospho-S2-
RNA Pol II binding appeared to be reduced more rapidly
within early downstream regions than general RNA Pol II
binding, suggesting that RNA Pol II dephosphorylation might
precede stalling and dissociation of the holoenzyme. In con-
trast, samples treated with TNF-? for 6 h (conditions allowing
for effective production of Tat) revealed strong association of
both general RNA Pol II as well as phospho-S2-RNA Pol II
along all regions of the HIV-1 DNA assessed. Thus, the pro-
gressive dephosphorylation of the CTD of RNA Pol II at
downstream regions observed during early TNF-? stimulation
may be responsible for the observed defect in Pol II elonga-
tion, and that this defect can be rescued by Tat.
CDK9 associates with downstream coding regions of actively
transcribed DNA, suggesting that CDK9 can transit with the
elongating RNA Pol II complex. To assess whether CDK9 is
associated with downstream HIV-1 transcription complexes,
ChIP analysis of CDK9 binding to HIV?5000 DNA in J-Lat
6.3 cells stimulated with TNF-? for various times was per-
formed. The kinetics of CDK9 association with HIV?5000
DNA (Fig. 6C) was quite similar to the patterns observed with
phospho-S2-RNA Pol II. Specifically, no appreciable down-
stream binding was detected until 4 h after TNF-? addition.
These findings indicate that the lack of downstream CDK9
binding coupled with the action of an “unopposed” phos-
phatase could underlie the lack of effective RNA Pol II elon-
gation occurring during the early phases of TNF-? stimulation.
HIV-1 transcriptional elongation is restricted by an OA-
sensitive phosphatase. CDK9 and phospho-S2-RNA Pol II
were absent from downstream regions of HIV-1 DNA during
early TNF-?-induced transcription, despite their effective
binding to proximal regions within the HIV-1 LTR. This ob-
servation raised the possibility that a cellular phosphatase de-
6050WILLIAMS ET AL. J. VIROL.
FIG. 6. NF-?B induces promoter-proximal, but not downstream, recruitment of CDK9 and serine-2 phosphorylation of RNA Pol II. (A) Early
and late TNF-?-induced HIV-1 mRNA transcript elongation is DRB sensitive. J-Lat 6.3 cells were either untreated or pretreated with DRB for
30 min and then stimulated with 20 ng/ml TNF-? for 1 or 6 h or left unstimulated. Total RNA was extracted and initiated, or elongated HIV-1
mRNA transcripts were quantitated by real-time RT-PCR (B). (C) TNF-? stimulation induces rapid recruitment of CDK9 and serine-2-
phosphorylated RNA Pol II to the HIV-1 LTR. Conversely, recruitment to downstream DNA is delayed. Fixed chromatin extracts prepared from
J-Lat 6.3 cells stimulated with 20 ng/ml TNF-? continuously for various times were immunoprecipitated with antibodies specific for serine-2-
phosphorylated RNA Pol II and assessed for enrichment in HIV-1 LTR DNA (left) or HIV?5000 DNA (right) by UV visualization of PCR
products in a gel stained with ethidium bromide. Data are representative of three independent experiments. Note the presence of serine-2-
phosphorylated RNA Pol II on the HIV-1 LTR in samples stimulated for 15 min; this association was markedly decreased when HIV?5000 DNA
was analyzed. Quantitation of enrichment (fold increase) above the no-antibody control is indicated below each sample. (D) RNA Pol II is
decreasingly associated with downstream regions of HIV-1 DNA in early, but not late, TNF-?-induced transcription of latent HIV-1. Samples
stimulated with TNF-? for 30 min and immunoprecipitated with RNA Pol II or phospho-S2-RNA Pol II antibodies were subjected to real-time
PCR quantitative analysis for the indicated HIV-1 DNA regions (E).
VOL. 81, 2007 EXPRESSION OF HIV-1 REQUIRES SUSTAINED NF-?B 6051
phosphorylates the elongating RNA Pol II complex, provided
that Tat has not been synthesized. We hypothesized that inhi-
bition of this phosphatase might promote effective elongation
during early TNF-?-stimulated transcription. OA is an effec-
tive inhibitor of RNA Pol II dephosphorylation by protein
phosphatase 1 (36). We tested the effect of OA on TNF-?-
induced expression of latent HIV-1 in J6.3?Red cells. OA
synergized with TNF-?, inducing HIV-GFP expression in
?35% of cells, compared to ?15% and ?1% in samples
treated with TNF-? or OA alone, respectively (Fig. 7A, left
panel). In contrast, no significant difference in ?B-DsRed2
expression was detected between samples treated with a com-
bination of TNF-? and OA or with TNF-? alone (Fig. 7A, right
To further confirm these effects of OA on TNF-?-induced
expression of latent HIV, initiated and elongated HIV-1
mRNAs were quantitated from J-Lat cells treated with OA
and/or TNF-? for 30 min. OA did not significantly enhance the
abundance of TNF-?-induced initiated HIV-1 mRNA (Fig.
7B, left panel), suggesting that the inhibitor was not acting by
enhancing transcriptional initiation. In contrast, cells treated
with OA and TNF-? expressed eightfold more elongated
HIV-1 mRNA than cells treated with TNF-? alone (Fig. 7B,
right panel). These data suggest that OA enhances latent
HIV-1 gene expression by promoting transcriptional elonga-
The augmentation of early TNF-?-induced HIV-1 mRNA
elongation by OA suggested that this inhibitor might promote
effective expression of latent HIV-1 in the absence of Tat. To
assess this possibility, J6.3?Red cells were nucleofected with
siRNA targeting Tat mRNA or a mismatched sequence and
cells were incubated with or without OA, stimulated with
TNF-? or left unstimulated, and assessed for HIV-GFP ex-
pression by flow cytometry 18 h later. Expression of HIV-GFP
was strongly blunted in siTat-treated cells stimulated with
TNF-? alone (Fig. 7C, left panel). In contrast, HIV-GFP ex-
pression was enhanced in siTat-treated samples incubated with
both OA and TNF-?. Of note, OA induced more expression of
HIV-GFP in siTatMM samples than siTat samples. Similar
results were obtained in J15.2?Red and J8.4?Red cells. Impor-
tantly, OA did not enhance general ?B-DsRed2 expression,
nor did it promote expression of Tat in siTat-treated cells (Fig.
7C, right panel, and data not shown).
To assess the mechanistic action of OA, the binding of RelA,
RNA Pol II, phospho-S2-RNA Pol II, and phospho-S5-RNA
Pol II (a marker of early transcriptional events ) to HIV-1
LTR and downstream DNA was assessed in OA-treated and
untreated cells stimulated with TNF-? for 30 min or 6 h. OA
did not significantly affect binding of RelA, Pol II, or either of
its phosphorylated forms to the HIV-1 LTR at either time
point (Fig. 7D, left panel). In contrast, binding of Pol II and
phospho-S2-Pol II to HIV?7000 DNA was enhanced by OA
treatment after 30 min of TNF-? stimulation (Fig. 7D, right
panel). No enhancement was observed in samples treated with
TNF-? for 6 h, indicating this effect was specific to early tran-
scriptional events. Induction of S5 phosphorylation of the CTD
was also observed at the LTR, in agreement with a prior report
(23). These findings suggest that an OA-sensitive phosphatase
progressively dephosphorylates the CTD of RNA Pol II in cells
treated for short periods with TNF-?. The absence of effective
recruitment of CDK9 to this elongating RNA Pol II complex
creates a situation where the action of this phosphatase is
To further delineate the role of NF-?B in the activation of
integrated but transcriptionally latent HIV-1 proviruses, we
performed kinetic analyses of RelA and the RNA polymerase
II interaction with the LTR in the J-Lat cellular model of
HIV-1 latency. We observed a dynamic bimodal pattern of
RNA Pol II recruitment to the LTR that paralleled and was
likely driven by the inherent oscillatory nature of nuclear NF-
?B. Two recent studies have similarly demonstrated that sus-
tained induction of the NF-?B signaling pathway produces
similar oscillations of RelA and RNA Pol II at host gene
promoters (7, 38), likely through the induction of NF-?B-
dependent expression of I?B? and A20, both potent inhibitors
of NF-?B. The pattern of RelA and RNA Pol II recruitment to
the HIV-1 LTR is coincident with the pattern of overall nu-
clear enrichment of RelA in response to a TNF-? stimulus,
implying that LTR binding reflects general NF-?B abundance.
Indeed, when NF-?B signaling was limited to a single round
by administration of TNF-? for a brief period of time, the
bimodal pattern of RelA and RNA polymerase II recruitment
to the latent HIV-1 LTR was lost.
Transient induction of NF-?B is sufficient to induce the
synthesis of many ?B-responsive genes, most notably I?B?
(35). Consequently, it was unexpected that expression of latent
HIV-1 required sustained rounds to NF-?B induction for ef-
ficient activation of the virus. To confirm that general ?B-
responsive gene expression is achieved with transient induction
of NF-?B, we constructed new Jurkat- and J-Lat-based cell
lines containing integrated ?B-DsRed2 reporter plasmids.
Analysis of these cell lines showed that transient induction of
NF-?B is sufficient for generalized ?B-dependent gene expres-
sion, but not for expression of latent HIV. These results
prompted us to analyze the mechanistic basis for the lack of
responsiveness by the HIV-1 LTR.
Analysis of HIV-1 mRNA transcript formation immediately
after induction of NF-?B revealed that, while initiated tran-
scripts readily accumulate, transcriptional elongation is im-
paired. Consistent with this observation, early studies of HIV-1
transcription described NF-?B as a strong inducer of transcrip-
tional initiation (28). However, more recent studies of NF-?B
in HIV-1 transcription have suggested a role in RNA Pol II
elongation as well as initiation (39). Low-level accumulation of
elongated HIV-1 mRNA transcripts is apparent immediately
after TNF-? stimulation; however, the rate of accumulation is
greatly reduced relative to the rate of initiated transcript syn-
thesis. During sustained stimulation with TNF-?, the rate of
elongated transcript formation begins to approximate the rate
of initiated transcript formation only at 4 to 6 h after induction.
Further, this delayed increase in RNA Pol II elongation is
dependent on de novo protein synthesis. These results raised
the possibility that the late increase in elongation reflects syn-
thesis of Tat, which through its recruitment of P-TEFb is
known to greatly increase elongation. We hypothesized that
the initial round of NF-?B recruitment to the HIV-1 LTR,
while inefficiently inducing elongation, is sufficient to produce
6052 WILLIAMS ET AL.J. VIROL.
small quantities of Tat. This newly synthesized Tat then syn-
ergizes with subsequent rounds of transcriptional initiation
induced by NF-?B to promote highly efficient RNA Pol II
elongation. Additionally, variations in Tat synthesis may un-
derlie the fractional response to TNF-? observed in J-Lat cells
(37). This hypothesis is supported by the finding that ectopic
expression of Tat drives robust expression of latent HIV-1 in
response to transient induction of NF-?B, suggesting that the
FIG. 7. OA rescues NF-?B induction of efficient expression of latent HIV. (A) OA synergizes with TNF-? to promote expression of latent HIV.
J6.3?Red cells were incubated with or without 30 nM OA for 1 h, and stimulated with 20 ng/ml TNF-? or left unstimulated, and HIV-GFP (left
panel) and kB-DsRed2 expression (right) was quantitated 18 h later by flow cytometry. (B) OA promotes early TNF-?-induced transcriptional
elongation. Total RNA was extracted from cells treated as for panel A, and initiated (left panel) and elongated (right panel) HIV-1 transcripts were
quantitated by real-time RT-PCR. Note that OA does not affect initiated transcript abundance but effectively promotes elongated HIV-1 mRNA
abundance. (C) OA promotes TNF-?-induced expression of latent HIV-1 in the absence of Tat. J6.3?Red cells were nucleofected with siRNA
directed against Tat mRNA or a mismatch sequence, treated with 30 nM OA or left untreated, and stimulated with TNF-? or left unstimulated,
and HIV-GFP (left) or ?B-DsRed2 (right) expression was assessed by flow cytometry. Note the rescue of HIV-GFP expression in Tat siRNA-
treated cells with OA and the absence of effect on ?B-DsRed2 expression. (D) OA promotes downstream association of P-S-RNA Pol II in early
transcription. J6.3?Red cells were pretreated for 1 h with 30 nM OA or left untreated and stimulated with TNF-? for 30 min, 1 h, or left untreated,
and ChIP assessment for RelA, RNA Pol II, P-S2-Pol II, and P-S5-Pol II was conducted at the HIV-1 LTR and HIV?7000 DNA by quantitative
PCR. Note the increase in Pol II and P-S2-Pol II enrichment in HIV?7000 DNA in samples treated for 30 min with TNF with OA versus without
VOL. 81, 2007EXPRESSION OF HIV-1 REQUIRES SUSTAINED NF-?B 6053
absence of this transactivator is key to the initial ineffectiveness
of RNA Pol II elongation. These conclusions also agree with a
recent computational model of the effects of Tat on HIV-1
gene expression (37).
The ability of NF-?B to induce transcriptional elongation
has been ascribed to its ability to recruit P-TEFb to sites of
transcription, the same RNA Pol II kinase partner employed
by Tat to induce elongation (2). Our studies with the P-TEFb
inhibitor DRB suggest that early TNF-? driven, NF-?B-depen-
dent transcriptional elongation involves this kinase complex.
ChIP analyses of CDK9, the kinase component of P-TEFb,
revealed a pattern of recruitment to the HIV-1 LTR coincident
with RelA. Assessment of functional phosphorylation of the
CTD of RNA Pol II in phospho-S2 RNA Pol II ChIP analyses
confirmed that the polymerase complex is similarly phosphor-
ylated in both early and late TNF-?-stimulated samples at
proximal sites on the HIV-1 LTR. In contrast, we observed
that the pattern of S2 RNA Pol II phosphorylation at more
distal regions of HIV-1 DNA was greatly reduced during NF-
?B-dependent versus Tat-dependent Pol II elongation. Simi-
larly, we observed that CDK9 is associated with downstream
HIV-1 DNA in late, but not early, NF-?B-driven transcription.
These findings raise the possibility that CDK9 transits with the
RNA Pol II complex in the context of Tat and reinforces S2
phosphorylation during elongation (Fig. 8). This is in agree-
ment with a recent study demonstrating Tat-induced rephos-
phorylation of RNA Pol II in vitro (5), as well as several other
studies demonstrating CDK9 association with coding regions
of actively transcribed host genes (15, 16, 20).
A recent study of HIV-1 latency reported by Kim et al. (23),
published during the preparation of the manuscript, similarly
demonstrated a minimally processive RNA Pol II complex in
TNF-?-stimulated infected Jurkat T cells in the absence of Tat.
Also consistent with our own work, this study demonstrates
increased binding of CDK9 to coding regions of HIV-1 DNA
in the presence of Tat. However, Kim et al. observed signifi-
cant RNA Pol II binding to the unstimulated latent HIV-1
LTR, while our ChIP studies consistently do not detect such
binding. Additionally, whereas we observe TNF-?-induced
binding of CDK9 to the HIV-1 LTR at early time points when
Tat is not present, Kim et al. did not observe appreciable
binding of CDK9 to the TNF-?-induced HIV-1 LTR in the
absence of Tat. These differences may be a function of the
experimental systems employed. Of note, the model of latency
employed by Kim et al. includes a significant level of active
HIV-1 expression in the unstimulated state (10% GFP-positive
cells). In contrast, very little basal expression occurs in the
J-Lat cell model, as indicated by ?1% GFP-positive cells. The
relatively high level of GFP under basal conditions reported by
Kim et al. could account for the presence of bound RNA Pol
II in the absence of stimulation. Finally, differences in the
experimental protocol for ChIP assays (absence of micrococcal
nuclease in the fragmentation step) might contribute to the
differences in the detection of CDK9 binding.
FIG. 8. Model for events influencing early and late TNF-?-induced HIV-1 transcription. (A) TNF-? stimulation of J-Lat cells induces
NF-?B-mediated recruitment of PTEF-b to the HIV LTR, which drives serine-2 phosphorylation of the CTD of coincidentally recruited RNA Pol
II. This phosphorylated polymerase is progressively dephosphorylated during elongation, limiting processivity. (B) Early, inefficient elongation
produces low levels of HIV-1 Tat, which recruits CDK9 to the HIV-1 LTR in a context capable of transiting with the elongating polymerase. This
continued association permits reinforcement of serine-2 phosphorylation of the CTD and prevents transcriptional stalling due to the action of CTD
6054WILLIAMS ET AL. J. VIROL.
We suspect that NF-?B-dependent RNA Pol II elongation is
inefficient because of the absence of CDK9 coupled with the
unopposed action of one or more phosphatases that progres-
sively dephosphorylate the CTD of RNA Pol II at downstream
DNA regions. These changes do not occur with Tat. Whether
Tat enhancement of RNA Pol II elongation is solely due to
effective recruitment of the CDK9 kinase or also involves ac-
tive inhibition of the key phosphatases attacking the CTD is
currently unclear. We further found a significant restoration of
TNF-?-induced expression of latent HIV-1 in the presence of
OA, an inhibitor of various phosphatases. These findings sug-
gest that a component of the restricting CTD phosphatase is
sensitive to OA. These findings support a model in which
NF-?B-dependent RNA Pol II elongation is greatly under-
mined by progressive dephosphorylation of the CTD. Prelim-
inary siRNA knockdown studies suggest that FCP1, a well-
characterized CTD phosphatase (6, 26), is not a key
component of this process. Future studies directed to identi-
fying the phosphatases limiting the processivity of RNA Pol II
will be of interest.
It is not yet clear whether latency as it exists in vivo is a
consequence of low-level viral gene expression or complete
suppression. We have chosen to evaluate cells displaying a
more complete suppression, as this highly restrictive environ-
ment would seem to prove most difficult to activate and purge.
These studies were conducted using the J-Lat cellular model of
HIV-1 latency, a model that though fulfilling many criteria for
HIV-1 latency bears an uncertain relationship to HIV-1 la-
tency occurring in infected patients. Analyses of NF-?B oscil-
lation within quiescent primary T cells activated by various
inducers have largely recapitulated the findings observed in the
J-Lat system (7, 38). Additionally, latent proviruses contained
in J-Lat cells are preferentially integrated into actively tran-
scribed genes, mirroring findings of infected resting CD4?T
cells in HIV-infected patients with effective suppression of
viremia (17, 25). Thus, we strongly suspect the biochemical
changes we have detected also occur in latently infected cells
The finding that sustained induction of NF-?B is required
for ?B-driven activation of HIV-1 latency has important im-
plications for therapeutic efforts to eliminate this viral reser-
voir. Potential agonists of latent HIV-1 should be designed that
take into account the importance of sustained induction of
NF-?B. Alternatively, these findings support the consideration
of combined application of soluble and cell-permeable forms
of Tat and transient agonists of NF-?B, or perhaps the elective
use of specific phosphatase inhibitors. However, given the
broad assortment of host genes induced by NF-?B and the
sensitivity of many gene products to phosphorylation-depen-
dent regulation, the combination of Tat and transient induc-
tion of NF-?B might avoid the toxicity likely to be associated
with sustained systemic induction of NF-?B or manipulation of
The authors thank G. Howard and S. Ordway for editorial assistance
and R. Givens for assistance with the preparation of the manuscript.
This work was supported by funds from NIH grant P01 AI058708,
the University of California San Francisco—Gladstone Institute of
Virology and Immunology Center for AIDS Research (UCSF-GIVI
CFAR) (P30 AI27763), and an NIH core equipment grant awarded to
the J. David Gladstone Institutes (RR1 892801).
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