Procyanidin trimer C1 derived from Theobroma cacao reactivates
latent human immunodeficiency virus type 1 provirus
Takanori Hori a, 1
, Jacob Barnor b, 1
, Tung Nguyen Huu c, 2
, Osamu Morinaga c
Akiko Hamano a
, Jerry Ndzinu a, b
, Angela Frimpong b
, Keren Minta-Asare b
Mildred Amoa-Bosompem b
, James Brandful b
, John Odoom b
, Joseph Bonney b
Isaac Tuffour b
, Baffour-Awuah Owusu b
, Mark Ofosuhene b
, Philip Atchoglo b
Maxwell Sakyiamah d
, Richard Adegle d
, Regina Appiah-Opong b
, William Ampofo b
Kwadwo Koram a
, Alexander Nyarko b
, Laud Okine d
, Dominic Edoh d
, Alfred Appiah d
Takuhiro Uto c
, Yoshiyuki Yoshinaka a
, Shin Uota a
, Yukihiro Shoyama c
, Shoji Yamaoka a, *
a Tokyo Medical and Dental University, Japan
b Noguchi Memorial Institute for Medical Research, Ghana
c Nagasaki International University, Japan
d Centre for Plant Medicine Research, Ghana
article info
Article history:
Received 30 January 2015
Available online 26 February 2015
Keywords:
HIV
Latency
Reactivation
Procyanidin
abstract
Despite remarkable advances in combination antiretroviral therapy (cART), human immunodeficiency
virus type 1 (HIV-1) infection remains incurable due to the incomplete elimination of the replicationcompetent virus, which persists in latent reservoirs. Strategies for targeting HIV reservoirs for eradication that involves reactivation of latent proviruses while protecting uninfected cells by cART are urgently
needed for cure of HIV infection. We screened medicinal plant extracts for compounds that could
reactivate the latent HIV-1 provirus and identified a procyanidin trimer C1 derived from Theobroma cacao
as a potent activator of the provirus in human T cells latently infected with HIV-1. This reactivation
largely depends on the NF-kB and MAPK signaling pathways because either overexpression of a superrepressor form of IkBa or pretreatment with a MEK inhibitor U0126 diminished provirus reactivation
by C1. A pan-PKC inhibitor significantly blocked the phorbol ester-induced but not the C1-induced HIV-1
reactivation. Although C1-induced viral gene expression persisted for as long as 48 h post-stimulation,
NF-kB-dependent transcription peaked at 12 h post-stimulation and then quickly declined, suggesting
Tat-mediated self-sustainment of HIV-1 expression. These results suggest that procyanidin C1 trimer is a
potential compound for reactivation of latent HIV-1 reservoirs.
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
Combination antiretroviral therapy (cART) can successfully
control the viral load of human immunodeficiency virus type 1
(HIV-1) in most HIV-1 infected patients and has led to a significant
decrease in the number of deaths related to acquired
immunodeficiency syndrome (AIDS) [1]. Although HIV-1 infection
is now managed as a chronic disease, persistence of HIV-1 in reservoirs, such as in resting memory CD4þ T cells, is a major obstacle
for HIV eradication [2]. Poor adherence to cART often permits the
emergence of mutant virus from reservoirs; therefore, new approaches to purge latent reservoirs and to discontinue cART (a
functional cure) are urgently needed. Such an approach is expected
to activate the latent provirus to promote death of latently infected
cells through viral cytopathic effects or killing by host cytotoxic T
lymphocytes (CTLs).
To reactivate HIV-1, a wide variety of approaches have been
proposed that employ histone deacetylase inhibitors (HDACIs),
methylation inhibitors, NF-kB activators, protein kinase C (PKC)
* Corresponding author. Department of Molecular Virology, Tokyo Medical and
Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan.
E-mail address: [email protected] (S. Yamaoka). 1 T.H. and J.B. contributed equally to this work. 2 Present address: School of Medicine and Pharmacy, Vietnam National University, Hanoi, 144 Xuan Thuy St., Cau Giay, Hanoi, Vietnam.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
http://dx.doi.org/10.1016/j.bbrc.2015.02.102
0006-291X/© 2015 Elsevier Inc. All rights reserved.
Biochemical and Biophysical Research Communications 459 (2015) 288e293
modulators, Akt/Hexim-1 modulators, and BET bromodomain inhibitors [3]. Clinical trials with HDACIs are underway, e.g., vorinostat (SAHA), panobinostat (LBH589), and romidepsin (FK288) [4].
PKC agonists that activate latent proviruses through NF-kB activation and are considered promising for killing of HIV-1 reservoirs
include prostratin and bryostatin, which have non-carcinogenic
properties and are capable of inducing HIV-1 transcription. A
clinical trial is in progress to determine whether bryostatin reduces
latent HIV-1 reservoirs. Plants are good source of structurally
diverse compounds and can therefore also serve as a potential
source of novel therapeutic agents. Here we describe a new class of
a plant-derived molecule that activates the latent HIV-1 provirus
through the ERK and NF-kB pathways in human T cells independently of PKC.
2. Material and methods
2.1. Cells
Jurkat-derived cell lines and ACH-2 cells were maintained in
complete RPMI 1640 medium supplemented with 10% fetal bovine
serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin.
HEK293T cells were propagated in Dulbecco’s modified Eagle’s
medium containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL
streptomycin.
2.2. Reagents
Tumor necrosis factor a (TNF-a) was purchased from Repro Tech
(Rocky Hill, NJ). Trichostatin A (TSA) was purchased from Sigma
Aldrich (St. Louis, MO), and a mitogen-activated protein kinase
(MEK) inhibitor U0126 was purchased from Promega (Madison,
WI). Pan-PKC inhibitor Go6983 was purchased from Calbiochem €
(San Diego, CA), and phorbol myristate acetate (PMA) was purchased from Enzo Life Sciences (Farmingdale, NY). Cells were
incubated with inhibitors for 1 h prior to activation. TNF-a was
resuspended in the RPMI medium, and other compounds were
resuspended in DMSO and stored as recommended by
manufacturers.
2.3. Plasmids
The lentiviral vectors capable of expressing an NF-kB-driven
firefly luciferase, CS-kB-R2.2 [5], and an EF1a-promoter-driven
Renilla luciferase, pCS-EF-1a-Rluc-puro (pCERp) [6] were described
previously. pNL4-3luc [7], the pCMV-DR8.2 packaging construct,
and a vector capable of expressing the vesicular stomatitis virus G
protein (VSV-G), pHCMV-VSV-G, were kind gifts from Dr. I. S. Y.
Chen (UCLA, Los Angeles, CA, USA). For construction of the pCSIIEF-SR-IB vector, the entire coding sequence of SR-IkBa was ampli-
fied by PCR from pMRX-SR-IkBa-GFP [8] using the following primer
set: Forward, 50
-CACCATGTTCCAGGCGGCCGA-30 and reverse, 50
TCATAACGTCAGACGCTGGCCT-30
. The PCR product was cloned into
the entry vector pENTR™/D-TOPO® (Invitrogen, Carlsbad, CA). The
SR-IkBa gene was transferred by recombination with the LR clonase
reaction (Invitrogen, Carlsbad, CA) into the lentiviral destination
vector pCSII-EF-IB-RfA [9]. The pCSII-EF-MCS-IB plasmid was used
as a control vector.
2.4. Preparation of virus stocks
The production of VSV-G-pseudotyped NL4-3luc and other
lentiviral vectors was described previously ([5,6,10]). The virus
supernatant was harvested at 48 and 60 h post-transfection and
was filtered through a 0.45 mm-pore syringe filter.
2.5. Generation of the JLR2 cell line and its derivatives
To generate reporter T cells latently infected with HIV-1, we
infected Jurkat cells with the VSV-G-pseudotyped NL4-3luc virus
and cultured cells until surviving cells proliferated sufficiently.
Single-cell clones were isolated by limiting dilution, and those
capable of inducing expression of the HIV-1 provirus in response to
PMA were selected. One such cell clone was transduced with the
pCERp lentiviral vector, and JLR2 cells were established. To establish
JLR2 cells expressing SR (JLR2 SR), JLR2 cells were infected with the
VSV-G-pseudotyped pCSII-EF-SR-IB lentiviral vector and selected
with 4 mg/mL of blasticidin S. Control cells (JLR2 EV) were established in a similar manner by transduction with the CSII-EF-MCS-IB
lentiviral vector. To establish JKR cells, Jurkat cells were infected
with VSV-G-pseudotyped CS-kB-R2.2 and pCERP lentiviral vectors
as described previously [6].
2.6. Extraction and isolation of the trimer procyanidin C1
Air-and-shade-dried cocoa beans (700 g) were milled and then
extracted with 50% aqueous EtOH (2.0 L, four times) under sonication at 40 C. The combined extracts were concentrated under
reduced atmospheric pressure yielding a crude residue (42.1 g),
which was defatted by partition between H2O and CHCl3 (500 mL,
three times). The aqueous fat-free cocoa extract (500 mL) was
subjected to a Diaion HP-20 column after washing with approximately 10 L of H2O. The column was then eluted by MeOH (2.0 L)
followed by evaporation and lyophilization, which yielded a deep
brown powder of total polyphenols (15.6 g). This powder (10.0 g)
was dissolved in MeOH (20 mL) and then applied to a 40 500 mm
column containing the Toyopearl HW-40F gel and eluted with the
same solvent to give 16 fractions (CC-1 through CC-16). Fraction CC-
6 was rich in trimers according to liquid chromatography with mass
spectrometry (LC-MS) analysis and was also found to exhibit
marked reactivation of the latent HIV-1 provirus. Therefore, fraction CC-6 was subjected to further chromatography on a reversedphase C18 column (YMC C18 ODS-A gel, 50 mm, 25 400 mm) with
MeOH:H2O (3:5, v/v) as the mobile phase followed by a Sephadex
LH-20 column (20 450 mm) with MeOH as an eluant. The last
procedure yielded 85 mg of trimeric procyanidin C1 (>95% purity
by HPLC). Nuclear magnetic resonance spectra and high-resolution
electrospray ionization time-of-flight mass spectrometry (HR-ESITOF MS) experiments were performed to identify procyanidin C1
using JEOL ECX 400 NMR and JEOL Accu TOF™ LC 1100 mass
spectrometers (JEOL, Tokyo, Japan).
Epicatechin-(4b/8)-epicatechin-(4b/8)-epicatechin (procyanidin C1) had the following characteristics: brown powder; Rf 0.42
[C18-TLC, MeOHeH2O (2:3, v/v)]; 13C-NMR (100 MHz, CD3OD) d 29.8
[C-4 bottom unit (b)], 37.4 [C-4 middle (m) and top (t) unit], 66.8 (C-
3 b), 72.9 (C-3 m), 73.5 (C-3 t), 77.0 (C-2 m and t), 79.7 (C-2 b), 96.2
(C-8 t), 96.6 (C-6 m and t), 97.6 (C-6 b), 100.6 (C-4a b), 102.3 (C-4a m
and t), 107.7 (C-8 b and m); B-ring carbons: 115.3 (C-20
), 116.0 (C-
50
t), 119.1 (C-60
), 132.1 (C-10
), 132.5 (C-10
), 144.3 (C-30
), 144.8 (C-30
145.6 (C-40
), and 145.8 (C-40
); A-ring carbons: 154.7, 156.6, 156.8,
157.3, and 157.9; positive HR-ESI-MS m/z 867.2154 [M þ H]þ
(calculated for C45H39O18, 867.2136).
2.7. Western blotting
Whole-cell lysates were prepared as described previously [10].
Approximately 30 mg of whole-cell lysates were subjected to SDSPAGE, and then the proteins were transferred to a polyvinylidene
difluoride (PVDF) membranes and analyzed by standard immunoblotting procedures using the following antibodies: antiphosphorylated-ERK1/2 (anti-p-ERK1/2) and anti-ERK1/2 (#9101
T. Hori et al. / Biochemical and Biophysical Research Communications 459 (2015) 288e293 289
and #9102, respectively, Cell Signaling Technology, Inc., Beverly,
MA), anti-IkBa (C-21, sc-371, Santa Cruz Biotechnology, Santa Cruz,
CA), and anti-HIV1 p24 [39/5.4A] anti-GAPDH (ab9071 and
ab8245, respectively, Abcam, Cambridge, England). The membranes were then incubated with a horseradish peroxidaseconjugated anti-rabbit or anti-mouse IgG secondary antibody
(#7074S, Cell Signaling Technology, Inc., Beverly, MA or A206PS,
American Qualex Antibodies, Inc., San Clemente, CA, respectively),
and proteins were visualized using the Immobilon Western
Chemiluminescent HRP Substrate (EMD Millipore Corporation,
Billerica, MA) or enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL).
2.8. The dual luciferase assay and an enzyme-linked
immunosorbent assay (ELISA)
Approximately 2.5 105 cells were stimulated, and then firefly
and Renilla luciferase activities were measured using the GloMAX
multidetection system (Promega Corp, Madison, WI) according to
the manufacturer’s instructions. The firefly luciferase activity was
normalized to Renilla luciferase activity. The amount of the Gag
protein in cell lysates was quantitated by an HIV-1 CA (p24) ELISA
kit (ZeptMetrix Corporation, Buffalo, NY).
3. Results
3.1. Screening of herbal extracts
One hundred and thirteen extracts were prepared using 50%
ethanol from 86 selected medicinal plants found in Ghana. The
extracts were used in the range of 3.125e100.000 mg/mL for
screening. To screen the selected Ghanaian medicinal plants for
compounds that could activate expression of the latent HIV-1
provirus, we established a subline of the Jurkat T-cell line as a
human T-cell latency model. HIV-1 gene expression can be
induced in latently infected cells by stimulation with phorbol esters, HDACIs, or cytokines, such as TNF-a. Treatment of JLR2 cells
with PMA, TSA, or TNF-a induced the expression of the proviral
reporter gene (Supplementary Fig. 1A). JLR2 cells were treated
with solutions containing 50 mg/mL of crude plant extracts dissolved in 50% ethanol. We determined the ratiometric expression
of the firefly luciferase relative to the Renilla luciferase after 20 h of
incubation. Ten of 113 extracts prepared from 86 plants reproducibly enhanced the viral reporter gene activity in JLR2 cells (data
not shown). In one of the screened plants, Theobroma cacao,
activity-guided fractionation by column chromatography procedures identified procyanidin trimer (Tc-1), tetramer (Tc-2), and
pentamer (Tc-3) as active compounds (Supplementary Fig. 2A).
Stimulation of JLR2 cells with Tc-1 induced expression of the HIV-
1-linked luciferase gene in a dose-dependent manner
(Supplementary Fig. 2B).
We selected Tc-1 for further experiments because it activated
the latent provirus at a lower concentration than did other oligomers. NMR studies revealed that the structure of Tc-1 is identical to
a previously reported compound C1[11] (Fig. 1A); thus, Tc-1 was
designated as C1 hereafter. Because the firefly luciferase gene was
inserted into the position of Nef of HIV-1, which is expressed from a
fully spliced HIV-1 mRNA, we considered the expression of p24 and
Pr55-Gag as another indicator of provirus stimulation from latency.
Immunoblotting experiments and ELISA showed that C1 induced
the expression of p24 or Pr55-Gag in JLR2 cells (Fig. 1C and
Supplementary Fig. 3A). To confirm that the reactivation of the
latent HIV-1 provirus with C1 is not a cell type-specific event,
similar experiments were performed on another human T-cell line
(ACH-2) that was latently infected with HIV-1 [12].
Immunoblotting experiments and ELISA showed that C1 induced
the expression of p24 or Pr55-Gag in ACH-2 cells (Fig. 1D and
Supplementary Fig. 3B).
3.2. NF-kB mediates the procyanidin C1-induced reactivation of
HIV-1
The long terminal repeat (LTR) of HIV-1 contains binding sites
for a variety of transcription factors, including NF-kB, NF-AT, AP-1,
and Sp1 [13]. Because C1 was recently reported to activate NF-kB
through phosphorylation of IkBa and translocation of RelA [11], we
analyzed the role of NF-kB in the reactivation of the latent HIV-1
provirus by C1. For this purpose, Jurkat cells were transduced
with two lentiviral vectors expressing firefly luciferase driven by an
NF-kB-dependent promoter and an EF1a promoter-driven Renilla
luciferase, and the resultant reporter cells were designated as JKR
cells. JKR and JLR2 cells were stimulated with PMA as a control or
with C1 to monitor the kinetics of NF-kB-driven reporter gene
expression or HIV-1 LTR-driven reporter gene expression. In JKR
cells, induction of the expression of the NF-kB-driven luciferase
reporter gene was apparent within 3 h after addition of C1 to the
culture medium and thereafter increased until it reached a peak
12 h after stimulation (Fig. 2C). This C1-induced NF-kB-dependent
transcriptional activity quickly decreased to baseline 36 h after
stimulation. PMA-stimulated cells showed robust induction of the
reporter gene expression, which reached a maximum at 12 h after
stimulation and remained high throughout the entire experimental
period. In JLR2 cells, the level of NL4-3 luciferase reporter gene
expression reached a maximum between 24 h and 36 h after
stimulation with C1 and remained high throughout the entire
experimental period. The kinetics of PMA-induced reporter gene
expression in JLR2 cells were similar to those caused by C1, but the
extent of gene induction by PMA was more than 10-fold greater
than that caused by C1. These findings provided evidence that
procyanidin C1 activated NF-kB quite transiently but caused
persistent activation of HIV-1 gene expression.
Next, to determine how NF-kB contributed to the production of
the latent provirus induced by C1, JLR2 cells were transduced with a
lentiviral vector expressing a super-repressor form of IkBa (SR),
which has mutations S32A and S36A rendering the molecule refractory to phosphorylation by IkB kinases, thereby specifically
suppressing NF-kB activation. In control vector-transduced JLR2
cells (JLR2 EV), PMA and C1 induced the HIV-1 gene expression as
expected (Fig. 2A). In contrast, cells expressing SR (JLR2 SR) failed to
substantially increase the reporter gene expression in response to
either C1 or PMA (Fig. 2A). Immunoblotting analyses revealed that
expression of HIV-1 Pr55-Gag and p24 was barely induced by either
C1 or PMA in the presence of SR in JLR2 cells (Fig. 2B). These
findings indicated that NF-kB activation played an important role in
the C1-mediated activation of the latent HIV provirus in JLR2 cells.
3.3. The MAPK pathway is involved in procyanidin C1-induced
provirus activation
C1 was reported to induce phosphorylation of ERK in mouse
macrophages [11]. To assess the importance of the MAPK pathway
in the C1-induced activation of the latent HIV-1 provirus in JLR2
cells, cells pretreated with the MEK inhibitor U0126 were stimulated with C1 or PMA, and the reporter luciferase activity was
measured. The U0126 treatment of JLR2 cells diminished phosphorylation of ERK induced by either C1 or PMA and profoundly
suppressed induction of the reporter gene expression (Fig. 3A and
B), suggesting that the MEK activation mediated the C1-induced
activation of the HIV-1 provirus in JLR2 cells.
290 T. Hori et al. / Biochemical and Biophysical Research Communications 459 (2015) 288e293
3.4. A pan-PKC inhibitor Go6983 does not inhibit C1-mediated €
provirus activation
We next tested whether PKC activation is involved in the C1-
mediated provirus reactivation. Pretreatment of JLR2 cells with a
pan-PKC inhibitor Go6983 potently inhibited the PMA-induced €
provirus gene expression in a concentration-dependent manner
but did not affect the C1-mediated provirus gene expression at the
highest concentration, indicating that C1 reactivated the latent
HIV-1 provirus independently of PKC (Fig. 3C). This notion was
further supported by the synergistic reactivation of the HIV-1
provirus by lower concentrations of C1 and PMA (Supplementary
Fig. 4A). C1 also synergistically worked with TSA in inducing the
provirus gene expression (Supplementary Fig. 4B). These results
reinforced the notion that reactivation of the HIV-1 provirus by C1
is mechanistically different from reactivation induced by PMA or an
HDAC inhibitor.
4. Discussion
Although various stimuli, including inflammatory cytokines,
phorbol esters, HDAC inhibitors, and PI-3 kinase agonists, have
been reported to reactivate the latent HIV-1 provirus, we report
here for the first time that a procyanidin trimer called C1 triggers
production of the latent HIV-1 provirus in human T cells. Our results
indicate that C1 activates the NF-kB and MAPK pathways, both of
which perform crucial functions in reactivation of the latent HIV-1
provirus by C1, and that PKC activity is not important for this C1-
mediated provirus activation (Fig. 4). Procyanidins have long been
implicated in the modulation of inflammatory responses, including
production of inflammatory mediators such as cytokines and eicosanoids as well as regulation of cellular signal transduction
pathways (see the review by Khan et al., 2014) [14]. For instance,
Hou et al. showed that proanthocyanidins with the galloyl moiety,
but not prodelphinidin without this moiety, inhibit cyclooxygenase
2 expression in lipopolysaccharide (LPS)-activated mouse macrophages by blocking the NF-kB and MAPK pathways (C1 lacking this
moiety was not tested) [15]. Mackenzie et al. reported that B
dimeric procyanidin inhibits PMA-induced NF-kB activation in
Jurkat cells [16]. The effects of the C1 trimer reported in this study,
which are apparently opposite to those of the B2 dimer in the Jurkat
cell line, may result from differences in the oligomerization status
of procyanidins. Terra et al. reported that procyanidins B1 dimer
and C1 trimer inhibit activation of IkB kinase b in LPS-stimulated
THP-1 cells pretreated with PMA for induction of differentiation
[17]. They also demonstrated inhibition of MAPK activation by
these procyanidins. It should be noted that THP-1 cells in that study
were pretreated with a high concentration of PMA for 24 h before
procyanidin treatment of THP-1 cells. It was reported that PMA
induces differentiation of THP-1 cells into macrophage-like cells,
where NF-kB activation is required for survival and differentiation.
Thus, it is conceivable that THP-1 cells that were pretreated with
PMA already have an elevated level of NF-kB activity when treated
with procyanidin B1 or C1, which may have resulted in a different
outcome in their study. Most recently, Sung et al. reported that C1
activates the NF-kB and MAPK pathways leading to production of
inflammatory cytokines in mouse macrophages [11]. Similarly, our
results clearly show that C1 reactivates latent HIV-1 proviruses in
human T cells via a mechanism dependent on the NF-kB and MAPK
pathways, although the cellular receptor mediating the C1-induced
activation of these pathways is yet to be identified.
The transient NF-kB activation and prolonged induction of viral
gene expression by C1 are noteworthy properties of this compound
because persistent NF-kB activation is likely to induce expression of
Fig. 1. Procyanidin C1 purified from Theobroma cacao activates the latent HIV-1 provirus. (A) Chemical structure of Tc-1 (C1). (B) JLR2 cells were stimulated with the indicated
concentrations of C1 or PMA. Luciferase activity was measured 20 h post-stimulation. The mean luciferase value in DMSO treated control cells was arbitrarily set to 1.0. The fold
induction and standard deviations in three independent experiments are shown. (C and D) JLR2 (C) or ACH-2 (D) cells were stimulated with the indicated concentrations of C1 or
PMA for 20 h. The Gag protein expression in JLR2 cells was detected by immunoblotting analysis with an anti-p24 or anti-GAPDH antibody.
T. Hori et al. / Biochemical and Biophysical Research Communications 459 (2015) 288e293 291
proinflammatory cytokines, an undesirable but likely effect of
provirus activators. As reported by Wolschendolf et al. for the HIV-
1-reactivating protein factor secreted by the nonpathogenic bacterium Massilia timonae [18], the C1-mediated transient NF-kB
activation is expected to initially produce a small but sufficient
amount of viral mRNA encoding Tat, which in turn greatly enhances
viral RNA production. This virus-specific regulatory mechanism is
believed to sustain a high level of viral gene expression in C1-
stimulated cells.
We detected synergistic activation of the HIV-1 provirus after
combined treatment of JLR2 cells with procyanidin C1 and a low
dose of either TSA or PMA. However, this synergistic reactivation is
not surprising because mechanisms of action of these three stimuli
are different from one another.
Fig. 2. Reactivation of the latent HIV-1 provirus by C1 requires NF-kB activation. (A) JLR2 cells transduced with the vector pCSII-EF-IB (JLR2 EV) or pCS-EF-SR-IB (JLR2 SR) were
stimulated with the indicated concentrations of C1 or 4 nM phorbol myristate acetate (PMA). Luciferase activity was measured 20 h post-stimulation. The mean luciferase value in
DMSO-treated control cells was arbitrarily set to 1.0. (B) Immunoblotting analysis of JLR2 EV or JLR2 SR cells stimulated with 25 or 50 mM C1 or 4 nM PMA. Approximately 30 mg of
each whole-cell lysate that was prepared 20 h after the stimulation was subjected to immunoblotting analyses using anti-p24, anti-IkBa, or anti-GAPDH antibodies. The data shown
are representative of three independent experiments. (C) Kinetics of HIV-1 LTR- or NF-kB-driven reporter gene expression after stimulation with either C1 or PMA. JLR2 (left) and
JKR (right) cells were incubated with DMSO (diamonds), 25 or 50 mM C1 (crosses and triangles, respectively), or 4 nM PMA (squares). Luciferase activity of each sample was
measured at the indicated time points. The mean luciferase activity level at time 0 was arbitrarily set to 1.0. Error bars indicate standard deviations in three independent
experiments.
Fig. 3. The MAPK inhibitor U0126 inhibits C1-mediated provirus activation, but the pan-PKC inhibitor Go6983 does not inhibit. (A) Immunoblotting analysis of JLR2 cells stimulated €
with the indicated concentrations of C1 or 4 nM PMA in the presence or absence of 10 mM U0126. The cells were harvested 2 h post-stimulation. Approximately 30 mg of each wholecell lysate was subjected to immunoblotting analyses using anti-pERK1/2 (pp44/42 MAPK) (Thr202/Tyr204), or anti-ERK1/2 (p44/42 MAPK) antibodies. (B) Luciferase activity in JLR2
cells stimulated with 25 or 50 mM C1 or 4 nM PMA in the presence or absence of 10 mM U0126 was measured 20 h post-stimulation. The mean luciferase value in DMSO-treated
control cells was arbitrarily set to 1.0. Error bars indicate standard deviations in three independent experiments. (C) JLR2 cells were pretreated with the indicated concentrations of
the pan-PKC inhibitor Go6983 1 h before stimulation with 25 or 50 € mM C1 or 4 nM PMA. In each condition, the mean luciferase activity level in DMSO-pretreated control cells was
arbitrarily set to 100. The mean luciferase values and standard deviations from three independent experiments are shown.
292 T. Hori et al. / Biochemical and Biophysical Research Communications 459 (2015) 288e293
In conclusion, we isolated and characterized procyanidin C1 as a
novel activator of the latent HIV-1 provirus. Although it remains to
be determined how C1 stimulates the NF-kB and MAPK signaling
pathways, the transient nature of NF-kB activation and sustained
induction of provirus gene expression are desirable properties of a
provirus-reactivating agent, dissociation of viral and cellular gene
induction.
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgments
This study was supported by the Science and Technology
Research Partnership for Sustainable Development (SATREPS)
Grant from Japan Science and Technology Agency and Japan International Cooperation Agency and in part by a grant-in-aid from
the MEXT Japan to S.Yamaoka (22117004-0 and 221S0001-02). We
thank Drs. Eiji Ido, Takao Masuda, Mitsuko Suzuki and Takashi
Suzuki, Mr. Sakae Kashihara, Mr. Yoshio Furuya and Mr. Yoshinobu
Sugiyama for their kind help and discussion.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.bbrc.2015.02.102.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2015.02.102.
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Fig. 4. Possible model of the C1-mediated reactivation of the latent HIV-1 provirus. C1
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provirus.
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