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IMMUNOBIOLOGY
Ectopic ATP synthase facilitates transfer of HIV-1 from antigen-presenting cells to
CD4⫹ target cells
*Amichai Yavlovich,1 *Mathias Viard,1,2 Ming Zhou,3 Timothy D. Veenstra,3 Ji Ming Wang,4 Wanghua Gong,2,4
Eliahu Heldman,1,2 Robert Blumenthal,1 and Yossef Raviv1,2
1Membrane
Structure and Function Section, Nanobiology Program, Center for Cancer Research, Bethesda, MD; 2Basic Research Program SAIC Frederick and
of Proteomics & Analytical Technologies, Advanced Technology Program, SAIC-Frederick, National Cancer Institute-Frederick, Frederick, MD; and
4Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, Bethesda, MD
3Laboratory
Antigen-presenting cells (APCs) act as
vehicles that transfer HIV to their target
CD4ⴙ cells through an intercellular junction, termed the virologic synapse. The
molecules that are involved in this process remain largely unidentified. In this
study, we used photoaffinity labeling and
a proteomic approach to identify new
proteins that facilitate HIV-1 transfer. We
identified ectopic mitochondrial ATP synthase as a factor that mediates HIV-1
transfer between APCs and CD4ⴙ target
cells. Monoclonal antibodies against the
␤-subunit of ATP synthase inhibited APCmediated transfer of multiple strains HIV-1
to CD4ⴙ target cells. Likewise, the specific inhibitors of ATPase, citreoviridin
and IF1, completely blocked APC-
mediated transfer of HIV-1 at the APCtarget cell interaction step. Confocal fluorescent microscopy showed localization
of extracellular ATP synthase at junctions
between APC and CD4ⴙ target cells. We
conclude that ectopic ATP synthase could
be an accessible molecular target for
inhibiting HIV-1 proliferation in vivo.
(Blood. 2012;120(6):1246-1253)
Introduction
Antigen-presenting cells (APCs), including dendritic and B cells,
play a major role in HIV pathogenesis.1,2 These cells act as vehicles
that transfer the virus to CD4⫹ lymphocytes, while simultaneously
activating these cells to produce high levels of HIV replication.
Using various imaging techniques, it has been shown that contact
between monocyte-derived dendritic cells (MDDCs) and T cells
facilitates transmission of HIV by locally concentrating virus,
receptor, and coreceptor at the intercellular adhesion point, forming
an infectious junction termed the virologic synapse.3,4 The cell-cell
transfer of HIV-1 involves binding and internalization by the donor
cell into intracellular compartments followed by release of virus
and transfer across the viral synapse to the target cell resulting in
infection. This phenomenon was extensively studied and reported
in some excellent reviews.1,5-7 The “virologic synapse,” which is
made of components of the immunologic synapse, explains the
high efficiency with which HIV-1 infects target cells by cell-cell
transfer. However, the mechanism of HIV internalization, synapse
formation, and cell-cell transmission is not known. Moreover, the
molecules within APCs and target cells that are involved in this
process remain largely unidentified. The DC specific intercellular
adhesion molecule grabbing nonintegrin (DC-SIGN) is the best
studied C-type lectin on the DC surface that captures HIV-1 and
transmits the virus to T cells.8-10 Nevertheless, DC-SIGN alone
cannot account for the multistep process of viral transfer, and the
possible involvement of other components has been proposed.9,11,12
In this work, we used a photoaffinity labeling and proteomic
approach to identify proteins that facilitate APC-mediated transfer
of HIV-1 to target cells. The ectopic ATP synthase was identified as
a factor that controls APC-mediated HIV-1 transfer at the intercellular level.
Submitted December 22, 2011; accepted June 24, 2012. Prepublished online
as Blood First Edition paper, July 2, 2012; DOI 10.1182/blood-2011-12-399063.
*A.Y. and M.V. contributed equally to this study.
The online version of this article contains a data supplement.
1246
Methods
Antibodies
Anti–DC-SIGN (clone DC-28) was a gift from Robert Doms from the
Department of Microbiology University of Pennsylvania School of Medicine. Anti-ATP synthase (2 clones, mouse monoclonal ab5432, Abcam; and
MS511, Mitoscience) and control mouse IgG Dye-conjugated antibodies
against monocyte and iDC markers were from BD Biosciences. Virus
isolates were produced from chronically infected cell lines as previously
described13 and were generously supplied by the AIDS and Cancer Virus
Program, SAIC Frederick Inc, Frederick MD. Pseudotyped HIV-Luciferase/
AD8 viruses were propagated in human embryonic kidney cells (293 cells).
Purified recombinant ATP synthase specific inhibitor IF1 was prepared by
“Varniss” (Frederick, MD). The TZM-bl indicator cell line,13 obtained
through the AIDS Research and Reference Reagent Program, Division of
AIDS, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, is a HeLa cell line derivative that expresses high levels
of CD4 and CCR5 along with endogenously expressed CXCR4. TZM-bl
cells contain HIV LTR-driven ␤-galactosidase and luciferase reporter
cassettes that are activated by HIV tat expression. DC-SIGN–expressing
Raji cells2 (DC-Raji) and HuT/CCR5 cells were generously provided by
Vineet KewalRamani, from the HIV drug resistance program National
Cancer Institute-Frederick, National Institutes of Health.
Monocyte derived DCs
The buffy coat fraction isolated from fresh donor blood was supplied by the
National Institutes of Health clinical center blood bank. Monocytes were
isolated by Percoll gradient centrifugation.14 Briefly, In a 50-mL conical
tube, the buffy coat fraction was overlaid on a layer of 15 mL Histopaque
(Sigma-Aldrich) and centrifuged for 30 minutes at 600g. The peripheral
blood mononuclear cell band at the interface between the plasma and the
histopaque was collected and washed 4 times in PBS and collected using
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2012 by The American Society of Hematology
BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
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BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
5-minute centrifugations at 500g. The peripheral blood mononuclear cells
were resuspended in 25 mL HEPES-buffered RPMI, and 5-mL fractions
were overlaid in 15-mL conical tubes on a 5-mL layer of 42% Percoll (GE
Healthcare) preequilibrated to isotonicity with 10 times PBS using an
osmometer. The cells were centrifuged for 30 minutes at 800g, and the
monocyte fractions at the interface between the RPMI and the percoll were
collected. The lymphocytes that separated in the pellet were collected as
well for some experiments. The monocytes were washed 3 times and
resuspended in RPMI 1640 ⫹ 10% FCS. The cells were incubated for
30 minutes at 37°C. The nonadherent cells were removed, and the adherent
monocytes were 93% positive for the CD14 marker as determined by flow
cytometry. Monocytes were differentiated to iDCs by incubating them at
1 ⫻ 106/mL in RPMI 1640 medium (RPMI 1640 ⫹ 10% FBS, 2mM
glutamine, 25mM HEPES, 100 U/mL penicillin, and 100 ␮g/mL streptomycin) in the presence of rhGM-CSF (50 ng/mL) and rhIL-4 (50 ng/mL) at
37°C in a humidified CO2 (5%) incubator for 7 days while changing the
medium every 48 hours. The resulting iDCs had the specific markers
DC-SIGN, CD11b, and CD11c determined by flow cytometry.
HIV-1 isolate induced labeling of DC-Raji cells surface proteins by
photoaffinity labeling. HIV-1 (MN) propagated in H9 T cells that included
approximately 50% (weight/weight) microvescicle proteins15 was reacted
with the trifunctional cross-linker sulfo-SBED (sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azidobenzamido)-hexanoamido)ethyl-1,3⬘dithioproprionate; Pierce Chemical, Thermo Fisher Scientific; supplemental Figure 1, available on the Blood Web site; see the Supplemental
Materials link at the top of the online article). The HIV-1 MN/H9
preparation (containing H9 cell microvesicles) in the amount of 0.35 mg
total protein (0.16 mg viral capsid, with a TCID50 of 3.2 ⫻ 105/mL) was
reacted with the succinimide ester moiety of sulfo-SBED in PBS in a
probe-to-protein molar ratio of 30:1. After 1-hour incubation, the reaction
was blocked by TBS and the virus was separated from the excess probe by
size exclusion chromatography on a PD-10 column. The modified virus
preparation was added to a total of 8 ⫻ 107 DC-Raji cells or MDDCs in
RPMI and incubated at 37°C for 3 hours by rotation. The cells were washed
twice with PBS and resuspended in 2 mL of the same buffer. The cells were
irradiated with UV light for 15 minutes to activate the phenyl azide moiety
and induce crosslinking between the bound virus and the proteins it binds to
on the cells. The light source was a 100-W ozone-free Mercury arc lamp
placed in a lamp house with collector lens. The cells were then washed and
lysed by freeze thawing in hypotonic buffer. The membrane fraction was
isolated by centrifugation of the lysate at 100 000g for 40 minutes and
collection of the pellet. Preliminary experiments have shown that practically all the biotin-labeled proteins were associated with the membrane
fraction of the cells.
Separation of biotinylated proteins by 2D electrophoresis
Proteins of the membrane fraction were separated by 2D electrophoresis
using membrane solubilization buffers that contained DTT to cleave the
S-S bond of the probe and thus deposit the biotinylated moiety on the target
protein. Isoelectric focusing buffer in the first dimension consisted of 7M
urea, 2M thiourea, 2% (weight/volume) amidosulfobetaine-14 (ASB14;
Calbiochem) and 0.5% (volume/volume) Triton X-100, 20mM DTT, and
0.5% (volume/volume) ampholytes. After incubation at 23°C for 2-4 hours,
bromophenol blue was added to 0.01% (weight/volume), and the sample
was centrifuged in a microcentrifuge at top speed for 10 minutes to remove
insoluble material. Samples were added to 11-cm pH 4-7 immobilized
pH gradient strips (GE Healthcare) or pH 3-10NL (nonlinear) immobilized
pH gradient strips (Bio-Rad). Immobilized pH gradient strips were
rehydrated with sample and focused for 35 000 volt-hours (Protean-IEF;
Bio-Rad). For the second dimension, the strips were equilibrated in SDS
buffer according to the manufacturer’s instructions and second dimension
SDS-PAGE was carried out followed by colloidal Coomassie or silver
staining. Two-dimensional separation of proteins was carried out in
different pH-ranges in the first dimension and 10% acrylamide in the second
dimension, with various gel sizes of 11, 18, and 24 cm. Gels were run in
duplicates; one was stained with colloidal Coomassie and the other was
transferred to nitrocellulose membrane for Western blotting. Biotinylated
proteins were detected either by enhanced chemiluminescence after stain-
ECTOPIC ATP SYNTHASE FACILITATES HIV-1 TRANSFER
1247
ing of the nitrocellulose membrane with streptavidin-peroxidase or by
infrared imaging on staining with streptavidin-alexafluor-680 (supplemental Figure 2C).
Separation of biotinylated proteins by affinity capture
To obtain the full spectrum of the proteins labeled by HIV in solution, an
extraction protocol was developed to solubilize all the biotinylated proteins
without creating a denaturizing condition that will interfere with the
subsequent affinity purification by streptavidin. The concentrated biotinlabeled membranes were solubilized in a minimal volume of 2% SDS
buffer, boiled for 3 minutes, and then diluted with 20 volumes of RIPA
buffer (50mM Tris-HCl, pH 7.4, 150mM NaCl, 0.25% deoxycholate,
1% NP-40, 1mM EDTA, and 100mM DTT). The final SDS concentration
was 0.1%, and the solution was centrifuged at 14 000g for 20 minutes for
removal of aggregates. A clear homogeneous solution was obtained without
the formation of precipitates. The complete solution of biotinylated proteins
was incubated with streptavidin-agarose overnight for affinity precipitation
of the biotinylated proteins. The agarose-streptavidin beads were washed,
and the biotinylated proteins were released by boiling the beads in SDS
buffer (2% SDS, 50mM DTT, and 62.5mM Tris-HCl, pH 8.0), and proteins
were identified by mass spectrometry (supplemental Figure 2B).
Identification of labeled proteins
Identification of isolated proteins was carried out by liquid chromatography
tandem mass spectrometry (LC-MS/MS) as previously described.16 Gel
bands stained with colloidal Coomassie were subjected to in-gel tryptic
digestion and the resultant peptides extracted. Each peptide sample was
desalted using C18 ZipTips as per the manufacturer’s directions (Millipore), lyophilized, and resuspended in 16 ␮L of 0.1% formic acid. Each
sample (6 ␮L) was loaded onto an Agilent 1100 nano-capillary HPLC
system (Agilent Technologies) equipped with a 10-cm integrated ␮RPLCelectrospray ionization emitter column (made in house), coupled online
to a LTQ XP mass spectrometer (Thermo Fisher Scientific) for ␮RPLCMS/MS analysis.
Measuring transfer of HIV-1 to target cells by APCs
DC-Raji cells or DCs (5 ⫻ 105/group) were resuspended in 100 ␮L PBS.
Different amounts of HIV-1, determined by a transfer assay calibration
curve, were added and brought to a final volume of 400 ␮L in RPMI for
each individual assay and then mixed with the cells and incubated at 37°C
for 3 hours with rotation. The virus concentration used was the one that
induced a transfer signal within the linear range as determined in separate
calibration transfer assays carried out for each individual HIV-1 strain and
lot (supplemental Figure 3). The APCs were washed twice in PBS and
resuspended in 500 ␮L/group of DMEM containing 40 ␮g/mL
diethylaminoethyl-dextran. The APCs were then added in triplicates to
96-well plates (150 ␮L/well) that were plated a day before with 2 ⫻ 104
TZM-bl cells/well. Infectivity of target TZM-bl was measured using the
luciferase reporter gene assay.2 After 24-hour incubation at 37°C, the
medium was aspirated and 50 ␮L of Steady-Glo Luciferase Assay substrate
was added to each well. After 20- to 30-minute incubation at room
temperature, the colorimetric signals were measured by a microplate
luminometer. In summary, the transfer assay consists of the following
consecutive steps: (1) mix APCs with HIV-1; (2) incubate at 37°C for
3 hours; (3) wash unbound virus twice and resuspend; (4) add the APCs to
TZM or HuT/CCR5 target cells; and (5) after coculture for 24 hours at
37°C, luciferase substrate was added and the resulting luminescence was
measured. For evaluating the effect of monoclonal antibodies on transfer
activity, 2 clones of anti-ATP synthase ␤-chain were added simultaneously
to enhance the effect to either the incubation step of HIV-1 and APCs (step
1) or to the step of incubation of the APCs with target cells (step 4). The
specified antibodies were applied each in the amount of 10 ␮g/reaction
(final concentration, 0.1 mg/mL). The ATP synthase inhibitors citreoviridin
and recombinant IF1 were added as specified for the individual experiments.
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1248
YAVLOVICH et al
BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
Confocal microscopy
MDDCs were incubated 1 hour (37°C) with Cell Tracker green chloromethylfluorescein diacetate (Invitrogen), and HUT/CCR5 cells were incubated
with Cell Tracker blue calcein AM at a dilution of 1:1000. After the
incubation with the dyes, the cells were resuspended in fresh medium and
washed with PBS. The MDDCs (5 ⫻ 105 cells) were incubated with
HIV-1–MN for 3 hours at 37°C, washed (2 times) with PBS, and added to
target TZM cells that were plated on poly-d-lysine–coated glass-bottom
dishes (35 mm, MatTek). When HUT/CCR5 cells were used as target cells,
the MDDC–HIV-1 were mixed with the target cells in a microcentrifuge
tube. Mouse anti-ATP synthase ␤-chain antibody (10 ␮g, Abcam; and
10 ␮g Mitoscience) was added at the time of mixing between MDDCs and
target cells. After incubation for 3 hours, the MDDC:HIV-1 plus target cell
complex was washed and fluorescently modified. Goat antimouse IgG
secondary antibody (1:2000, AlexaFlour-555; Invitrogen) was added for
1 hour (4°C). The samples were fixed with formaldehyde (4%, 15 minutes,
25°C) and then washed again with PBS. Image acquisition was carried out
using a Zeiss LSM 510 and LSM710 (Carl Zeiss) confocal laser scanning
microscope. A 100⫻/1.3 NA oil immersion objective lens was used with a
zoom factor of 4.0. Ar⫹ laser line (488 nm) was used for Cell Tracker green.
Chloromethylfluorescein diacetate excitation and emission light were
collected with a 500-550 bandpass filter. AlexaFluor-555 was excited by
HeNe 543-nm laser line (HFT UV/488/543/633, Mirror NFT 545) and
emission light was collected by 565-615 bandpass filter. Data were
corrected with background subtraction and analyzed by Zeiss LSM
Image Version 4.2 software.
Results
Identification of proteins labeled by HIV-1 induced photoaffinity
labeling of DC-Raji cellular proteins
Photoaffinity labeling has been applied to identify receptors for
protein ligands that were modified with a photoactivatable crosslinker. In this work, the photoactivatable crosslinker (SBED) was
bound to the surface proteins of HIV-1 viruses and membrane
vesicles derived from T cells that react with APCs in the context of
viral transfer and the formation of the virologic synapse. The
photoinduced crosslinked proteins on DC-Raji cells and immature
MDDCs that became labeled with biotin were separated and
subjected to mass spectrometry (MS) analysis. Two different
methods for separating the biotinylated proteins were used (Figure
1): (1) Two-dimensional polyacrylamide gel electrophoresis
(2D-PAGE) of the bulk membrane fraction followed by protein
transfer to nitrocellulose membranes and affinity staining of the
membrane with streptavidin conjugated to AlexaFluor and/or HRP;
and (2) sodium dodecyl sulfate (SDS)–assisted total solubilization
of the biotinylated proteins followed by affinity capture of the
biotinylated proteins using streptavidin agarose beads. The captured proteins were released by boiling in SDS and separated by
one-dimensional SDS-PAGE. Ten biotinylated protein spots were
detected on the 2D-PAGE gel in the pI and molecular weight ranges
of 5.5 and 45-60 kDa, respectively. These spots were excised and
identified using LC-MS/MS. Likewise, 10 stained proteins from
the affinity-captured proteins were also excised and analyzed. The
identified proteins obtained from 2 different APCs (DC-Raji and
monocyte-derived immature DCs) were selected based on the
number of peptides identified for each and their presence within
4 replicative experiments. One major protein, ATP synthase
␤-chain, stood out in this analysis (Figure 1B-C).
Figure 1. Composite figure describing the different fractions isolated and
analyzed after photoaffinity labeling. (A) After photoaffinity labeling, DC-Raji cells
were lysed and the membrane fraction (M) was isolated from the cytosolic fraction
(S) by centrifugation as described in “Methods” for separation of proteins. Samples
from both fractions were subjected to Western affinity staining with streptavidin-HRP
for the identification of the biotin-containing fraction. (B) The membrane fraction of
APC was solubilized in solubilization buffer, and the biotinylated proteins were
isolated by affinity capture using streptavidin-agarose beads. Ten bands were
subjected to digestion/tandem MS identification. An arrow indicates the identified
band containing ATP synthase. The distribution of the peptides was as follows: DC-Raji
cells (2 bands): band 1, 18 peptides of ATP synthase ␤-chain, 10 unique 15 peptides of ATP
synthase ␣-chain, 9 unique. Altogether, 12 unique peptides of ATP synthase ␤-chain
(19 total). Band 2, 9 peptides of ATP synthase ␤-chain, 9 unique, 6 peptides of ATP
synthase ␣-chain, 5 unique. Altogether, 9 unique peptides of ATP synthase ␣-chain
(14 total). DCs (2 bands): band 1, 38 peptides of ATP synthase ␤-chain, 15 unique
11 peptides of ATP synthase ␣-chain, 8 unique. Altogether, 14 unique peptides of
ATP synthase ␤-chain (43 total). Band 2, 5 peptides of ATP synthase ␤-chain,
3 unique 24 peptides of ATP synthase ␣-chain, 14 unique. Altogether, 15 unique
peptides of ATP synthase ␣-chain (35 total). Vimentin was also highly represented in
those DC samples. (C) The membrane fraction was solubilized and subjected to 2D
electrophoresis as described in “Methods” for separation of proteins. ATP synthase
was identified in the spots indicated by an arrow. Peptides recovered (4 spots):
519 peptides of ATP synthase ␤-chain, 24 unique.
The effect of anti-ATP synthase ␤-chain antibodies on
APC-mediated transfer of HIV-MN
To confirm the involvement of ATP synthase in HIV-1 transfer,
monoclonal antibodies against ATP synthase were added to DCRaji or MDDC at different stages of the transfer assay as described
in “Measuring transfer of HIV-1 to target cells by APCs.” Anti-ATP
synthase antibodies had no effect on the transfer activity when
added during incubation of the virus with APC (Figure 2A). In
contrast, the anti-DC-SIGN antibody had a profound inhibitory
effect when added at this step (Figure 2B). Because both the virus
and the microvesicles are derived from T cells, ATP synthase
␤-chain may bind to a T cell–associated ligand involved in the
interaction of the APC with the target cells through the formation of
the virologic synapse. To examine this possibility, APCs (DC-Raji
and MDDCs) were incubated with the virus, washed, and mixed
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BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
ECTOPIC ATP SYNTHASE FACILITATES HIV-1 TRANSFER
1249
Figure 2. Anti-ATP synthase antibodies block APC-mediated transfer of HIV-1 at the cell-cell interaction step. APC-mediated HIV-1 transfer was carried out either with
DC Raji cells (A-B) or with DCs (C-D). The indicated antibodies (20 ␮g/mL) were added either at the step of APC–HIV-1 interaction (A,C) or added at the step of incubation of
the virus-primed APCs with target TZM cells (B,D). No Ab indicates no antibody added; IgG, nonimmune mouse IgG; DC-SIGN, anti–DC-SIGN; and anti-ATPsyn, anti-ATP
synthase ␤-chain. All antibodies were at a concentration of 1.2 ⫻ 10⫺7M. Transfer activity was measured by viral infectivity given in luminescence arbitrary units. A total of 100%
transfer activity was determined as the maximal activity obtained in the absence of added antibody (No Ab). Experiments were carried out at least 3 times, and bar graphs
represent SEM of triplicate samples.
with the target cells (TZM) in the presence of anti-ATP synthase
antibody. As seen in Figure 2B and D, the anti-ATP synthase
antibody had a profound inhibitory effect on viral transfer to the
target cells, whereas the anti–DC-SIGN antibody had no effect.
Similar results were obtained using pseudotyped viruses with the
T-cell line Hut/CCR5 as target cells. Control experiments were
carried out for testing possible inhibitory effects that the antibodies
may have on HIV-1 infectivity of target cells (TZM), which is the
basis of our transfer assay. No significant effect was detected on
HIV-1 infectivity by any antibodies used in this study.
The inhibitory effect of anti-ATP synthase ␤-chain antibody on
DC-mediated transfer of HIV-1 is not affected by the cell line in
which the viruses were propagated
We next tested whether the role that ATP synthase has in HIV-1
transfer is dependent on the cell line in which the viruses were
grown. Many cellular proteins have been identified that adhere to
HIV-1 virions after budding,17,18 and these may play a role in the
Anti-ATP synthase ␤-chain monoclonal antibodies inhibit DCs
mediated transfer of various strains of HIV-1 at the step of the
virologic synapse formation
To evaluate whether the role of ATP synthase ␤-chain in the
DC-mediated transfer of HIV-1 is general and common to other
strains of HIV-1, MDDC-mediated HIV transfer was measured
using different HIV-1 strains (BAL, ADA, SF162, NL4-3, and MN)
that were propagated in one T-cell line (SUP-T1). The effect of the
anti-ATP synthase antibody was measured for each strain. The
control antibody, anti-DC-SIGN, had no significant effect on the
transfer of any of the HIV-1 strains tested. Nonimmune mouse IgG
was used as a negative control. Figure 3 conclusively shows that
the antibody against ATP synthase ␤-chain blocks the MDDCmediated transfer of all tested HIV-1 strains, indicating a general
role for ATP synthase in the transfer of HIV-1 mediated by primary
DCs at the cell-cell interaction step.
Figure 3. Anti-ATP synthase ␤-chain antibodies block DC-mediated transfer of
multiple strains of HIV-1. The indicated HIV-1 strains were all propagated in the
Sup-T1 cell line. Data are presented as in Figure 1. All antibodies used in this figure
as well as in subsequent figures were at concentrations as specified in “Measuring
transfer of HIV-1 to target cells by APCs” and for Figure 1.
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YAVLOVICH et al
BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
Specific inhibitors of ATP synthase activity block
HIV-1–mediated transfer by DCs
We next determined whether the activity of ATP synthase plays a
role in the MDDC-mediated transfer of HIV-1 to target cells. Two
specific inhibitors of the mitochondrial ATP synthase, citreoviridin
a potent inhibitor produced by fungus,19 and the endogenous
specific inhibitor IF120 were applied at the virologic synapse step.
Citreoveridin completely inhibited virus transfer at concentrations
levels where the toxin had no effect on cell viability or infectivity
of the of HIV-1 (Figure 4A). Likewise, the endogenous inhibitor of
ATP synthase, IF1, blocked APC-mediated transfer of HIV-1 in a
dose-dependent manner at pH 6.5, the optimum pH for IF1 specific
inhibition of ATP synthase in the mitochondria21 (Figure 5B-C).
Figure 4. The inhibitory effect of anti-ATP synthase ␤-chain antibodies on HIV-1
transfer is not affected by the cell line in which the virus is propagated. HIV-1
MN was propagated in the different T-cell lines as indicated. MDDC-mediated viral
transfer activity to target TZM cells was measured as described in “Measuring
transfer of HIV-1 to target cells by APCs.” A total of 100% transfer activity was
determined for each cell line as the maximal transfer activity obtained in the absence
of added antibody (No Ab).
biology of the virus. For that purpose, anti-ATP synthase antibodies
were tested for their ability to inhibit transfer of HIV-1 MN that
was propagated in different cell lines. The T-cell lines examined
were H9, SUPT1, Jurkat-TAT-CCR5, CEMX174, and H9-CL.4.
Experiments were carried out essentially as described in Figure
2. The results presented in Figure 4 show that the antibody inhibits
the MDDC-mediated transfer of HIV-1 from all tested T-cell lines.
Likewise, anti-ATP synthase antibody also blocked the transfer
of pseudotyped HIV-1 AD8 viruses that were expressed and
propagated in 293 cells while using the T-cell line HuT/CCR5 as
target cells.
HIV-1 induces the localization of ATP synthase ␤-chain at the
junction of APC and target cells
We wished to establish whether ATP synthase undergoes an
HIV-1–assisted localization and sequestration at the APC-target
cell junction, as implied by the biochemical data. An immunofluorescence experiment was carried out in which MDDCs were first
incubated with HIV-1, washed, and then added and incubated with
target cells (TZM or HUT/CCR5) in the presence of anti-ATP
synthase antibody. For fluorescence microscopy measurements,
APC and target cells were stained with distinct flluorescent probes
and ATP synthase was visualized using a fluorescent anti–mouse
IgG secondary antibody as described in “Confocal microscopy.”
The cells were fixed, and fluorescence was monitored with a
confocal microscope. The images presented in Figure 6 show
that ATP synthase is concentrated at the junction between
HIV-1–primed APCs and target cells.
Figure 5. ATP synthase inhibitors prevent APCmediated HIV-1 transfer at the cell-cell interaction
step. (A) The ATP synthase inhibitor, citreoviridin, was
added to the transfer assay with DCs at the indicated
concentrations either at step 1 (diamonds) or at step
4 (triangles) of the assay as described in “Measuring
transfer of HIV-1 to target cells by APCs.” “Infectivity”
represents the direct effect of the inhibitor on the infectivity of the virus as measured directly on TZM cells. The
infectivity in the absence of citreoviridin was determined
as 100%. (B) Dose-dependent inhibition of APC-mediated
HIV-1 transfer by recombinant IF1 at different pH. Viral
transfer was mediated by DC-Raji cells, and 25 ␮g
(8.3 ⫻ 10⫺5M) of the endogenous inhibitor IF1 was added
at the step of APC-target cell interaction. (C) Effect of
different inhibitors on DC-mediated HIV-1 transfer at the
step of APC-target cell interaction. Control indicates no
inhibitors. The amount of protein used per assay was
10 ␮g (1.3 ⫻ 10⫺7M) for each antibody and 25 ␮g
(8.3 ⫻ 10⫺5) for IF1. Experiments were repeated at least
3 times and were performed in triplicates. Bars represent
the SEM of the individual samples in a group.
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BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
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Figure 6. Immunocytolocalization of ATP synthase at
the junction between HIV-1–primed DCs and target
cells. MDDCs were incubated with HIV-1 for 3 hours,
washed, and added to target cells either TZM cells (A) or
HUT/CCR5 cells (B,D) in the presence of anti-ATP
synthase ␤-chain antibodies. (C) Same as in panel
A, only that the incubation was done without the virus.
(D) Same as in panel B, only that MDDCs were stained in
green and HUT/CCR5 were stained in blue as described
in “Confocal microscopy.” For all samples, staining was
carried out with Alexa-555–anti–mouse IgG secondary
antibody, the sample was fixed, and the pattern was
visualized by confocal microscopy as described in
“Methods.”
Discussion
Mediator cells, such as APC, which bind HIV-1 and transfer the
virus to its target CD4⫹ cells, are a major factor in the process of
virus dissemination and infection in vivo. The discovery of the cell
surface proteins that facilitate virus transfer can provide insights
into the formation of the virologic synapse between the APC and
the target T cell and identify potential molecular targets for
intervention in this process. In this study, a proteomics strategy
combining photoaffinity labeling and subfractionation was used to
discover proteins on the APC that facilitate transfer of HIV-1. The
crosslinker (SBED) was placed on the HIV-1 MN isolate from
H9 T cells that consisted of a mixture of HIV particles and cellular
microvesicles.15 Aside from the viral envelope proteins present
exclusively of HIV, the vesicles and viruses both contain T cell
ligands (eg, LFA-1 and ICAM-1) that are known to participate in
the formation of the immunologic or virologic synapse.6,7,17,18 After
crosslinking, the ␤-subunit of ATP synthase was consistently
identified on both DC Raji cells and MDDCs as a dominant component
in multiple targeted photolabeling experiments (Figure 1).
It is important to note that labeling of these proteins on DC-Raji
cells was triggered at the end of a 3-hour incubation period at 37°C
of a mixture of the virus and cells (step 3; see “Measuring transfer
of HIV-1 to target cells by APCs”); therefore, any labeled protein
may not necessarily represent the initial binding sites of the virus
but rather those that the virus interacts with after some cellular
trafficking, internalization, or endocytosis has taken place. Three
main approaches were used to establish the relevance of the
identified protein to the APC-mediated transfer of HIV-1:
(1) measuring the effect of monoclonal antibodies against ATP
synthase ␤-chain on HIV-1 transfer by DC-Raji cells and DCs;
(2) evaluating the effect of specific inhibitors of ATP synthase
activity on viral transfer by MDDC; and (3) cytoimmunolocalization of ATP synthase in the context of viral transfer. Antibodies
against ATP synthase ␤-chain almost completely block viral
transfer but only when added to the intercellular interaction step
between the virus primed APC and target cell (TZM or HuT/CCR5;
Figure 2B,D). The antibody had no or little effect when added at the
initial step of interaction between the APC and the virus (Figure
2A,C). This finding indicates that the ligand or effector molecule
that induced the photolabeling of ATP synthase ␤-chain on APC is
probably a T-cell component that represents a target cell (T cell)
contribution to the formation of the virologic synapse. This
T cell–associated ligand may have come either from the T cell–derived
microvesicles or the virus. The envelope proteins of the virus most
probably did not contribute to the photolabeling, which is consistent with the finding that DC-SIGN was not present among the
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1252
BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
YAVLOVICH et al
photolabeled proteins. This result is not entirely surprising because
the heavily glycosylated envelope proteins gp-120/gp41 will not
readily react with the succinimide moiety of SBED, which is
selective toward amino side chains in proteins.
Anti-ATP synthase antibodies completely blocked transfer of
multiple strains of HIV-1, indicating that the role of ATP synthase
in the APC-mediated transfer of HIV-1 is a general phenomenon
common to many strains of HIV-1 (Figure 3). Further support for
this conclusion comes from the finding that the T-cell line in which
the virus was grown had no effect on the inhibition of transfer
induced by anti-ATP synthase antibody (Figure 4). HIV-1 transfer
activity seems to be dependent on the enzymatic activity of ATP
synthase because the specific inhibitors of this enzyme, the fungus
derived cytreoviridin and the human endogenous inhibitor of
mitochondrial F1F0 ATP synthase, IF1, block APC-mediated viral
transfer at the cell-cell interaction step (Figure 5). The inhibitory
effect of IF1 is conferred by specific binding of this protein to the
F1F0 ATP synthase. The experiments carried out using antibodies or
inhibitors were controlled by showing that those had no effect
neither on the viability of the cells involved (APC or TZM) or on
the infectivity of the virus as measured in target cells by the
luciferase reporter gene assay (supplemental Figures 3 and 4).
Immunocytolocalization of extracellular ATP synthase ␤-chain
shows that this protein is localized at junctions between DCs and
target cells after priming of the DCs with HIV-1 (Figure 6). The
immonolocalization was done on intact cells without permeabilizing agents, indicating that the binding of the antibodies was to an
extracellular component of the enzyme. The localization of extracellular ATP synthase at the APC-target cell junction is consistent
with the premise that the enzyme participates in the transfer of the
virus from the APC to the CD4⫹ target cells. Localization of the
virus at that junction has been previously demonstrated as the site
for virus transfer.3-5 Our finding that ATP synthase is found at the
junctions between APC and CD4⫹ target cells, even without the
presence of the virus (Figure 5C), suggests that the virus is only
using the enzyme at this location as a facilitator of the transfer. ATP
sythase activity seems to be vital for this process as inhibitors of the
ATPase component of ATP synthase, citreoviridin and IF1, inhibited HIV transfer from APC to CD4⫹ target cells. The involvement
of ATP synthase ␤-chain in APC-mediated HIV-1 transfer and the
sequestration of this enzyme at the junction of APC and target cells
are not entirely surprising. Energy demanding remodeling of cell
cytoskeletal proteins, like actin, has been shown to be essential for
the formation of structural elements of the immunologic and
virologic synapse.22 Previous studies have demonstrated the dynamic nature of the mitochondria that undergo migration and
translocation to cellular “hot spots” where ATP is in high demand,
including the immunologic and neurologic synapse.22-26 A more
detailed structural study of the virologic synapse at the junction of
dendritic and T cells shows membrane remodeling and cytoskeletal
conduits that form by the contact between the 2 cells through which
the virus migrates from one cell type to another.27 This process may
also be triggered by T-cell antigens present on membrane microvesicles and/or HIV-1 particles leading to photoaffinity labeling
of ATP synthase ␤-chain. Previous studies have shown that this
protein, as part of the whole and active F1/F0 complex, is present
on the surface of endothelial cells, tumor cells,28-30 and multiple
cell lines.31-34 ATP synthase is a protein present on the inner
membrane of the mitochondria. The mechanism by which it
migrates to the surface of the cell is not understood. The significance of the topologic orientation of this enzyme as it pertains to
this study is that, contrary to anti–HIV-1–neutralizing antibodies
that do not block viral infection when applied at the virologic
synapse,35,36 anti-ATP synthase ␤-chain antibody completely blocks
APC-mediated infection of target cells at that step. This finding
makes ATP synthase an attractive and accessible target for
blocking the APC-mediated infection of HIV-1.
Acknowledgments
The authors thank Dr Vineet KewalRamani from the HIV drug
resistance program National Cancer Institute-Frederick, National
Institutes of Health; Julian Bess from the AIDS and Cancer virus
program SAIC-Frederick Inc for HIV isolates and expert advice;
Steven Lockett and Kim Peifley from the Optical Microscopy and
Analysis Laboratory SAIC-Frederick for expert imaging assistance; and Dr Michael F. Marusich, from Mitoscience Inc for
material support and expert advice on ATP synthase and oxidative
phosphorylation.
This work was supported in whole or in part by the Frederick
National Laboratory for Cancer Research, Intramural Research
Program of the National Institutes of Health (contract
HHSN26120080001E) and the Intramural AIDS Targeted Antiviral
Program.
The content of this publication does not necessarily reflect the
views or policies of the Department of Health and Human Services,
nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
Authorship
Contribution: M.V., R.B., and Y.R. designed research; A.Y., M.V.,
Y.R., and E.H. performed research; J.M.W. and W.G. contributed
new reagents; M.Z. and T.D.V. analyzed data; Y.R. wrote the
manuscript; and M.V., R.B., and E.H. edited the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Yossef Raviv, Frederick National Laboratory
for Cancer Research, Bldg 469, Room 213, PO Box B, Frederick,
MD 21702; e-mail: [email protected].
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2012 120: 1246-1253
doi:10.1182/blood-2011-12-399063 originally published
online July 2, 2012
Ectopic ATP synthase facilitates transfer of HIV-1 from
antigen-presenting cells to CD4 + target cells
Amichai Yavlovich, Mathias Viard, Ming Zhou, Timothy D. Veenstra, Ji Ming Wang, Wanghua Gong,
Eliahu Heldman, Robert Blumenthal and Yossef Raviv
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