SDF-1/CXCL12 production by mature dendritic cells inhibits the propagation of X4-tropic HIV-1 isolates at the dendritic cell-T-cell infectious synapse.
ABSTRACT An efficient mode of HIV-1 infection of CD4 lymphocytes occurs in the context of infectious synapses, where dendritic cells (DCs) enhance HIV-1 transmission to lymphocytes. Emergence of CXCR4-using (X4) HIV-1 strains occurs late in the course of HIV-1 infection, suggesting that a selective pressure suppresses the switch from CCR5 (R5) to X4 tropism. We postulated that SDF-1/CXCL12 chemokine production by DCs could be involved in this process. We observed CXCL12 expression by DCs in vivo in the parafollicular compartment of lymph nodes. The role of mature monocyte-derived dendritic cells (mMDDCs) in transmitting R5 and X4 HIV-1 strains to autologous lymphocytes was studied using an in vitro infection system. Using this model, we observed a strong enhancement of lymphocyte infection with R5, but not with X4, viruses. This lack of DC-mediated enhancement in the propagation of X4 viruses was proportional to CXCL12 production by mMDDCs. When CXCL12 activity was inhibited with specific neutralizing antibodies or small interfering RNAs (siRNAs), the block to mMDDC transfer of X4 viruses to lymphocytes was removed. These results suggest that CXCL12 production by DCs resident in lymph nodes represents an antiviral mechanism in the context of the infectious synapse that could account for the delayed appearance of X4 viruses.
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ABSTRACT: Dendritic cells and their subsets are among the very first immune cells to tackle incoming pathogens and initiate innate and adaptive immune responses. During the past year, some studies investigating the early events occurring at mucosal sites, upon HIV infection, reinforced our view that the virus has evolved subtle strategies to hijack key cellular components in dendritic cells, thus leading to viral acquisition and dissemination while dampening or delaying antiviral responses. In this review, we will detail recent research aimed at investigating the involvement of different dendritic cell subtypes on HIV transmission at mucosal sites, the events and cellular factors in dendritic cell guiding HIV trafficking, and polarization at the virological synapse. Furthermore, we will link some of these basic findings with current and novel therapeutic and prophylactic strategies targeting the early events of mucosal HIV transmission. It is becoming evident that a better characterization of the early events of HIV transmission and the involvement of dendritic cell subtypes in this process would contribute to strengthen our efforts to improve the current therapeutic and prophylactic strategies.Current opinion in HIV and AIDS 09/2011; 6(5):391-7. · 4.75 Impact Factor
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ABSTRACT: Concurrent chemotherapy and radiotherapy (CRT) has become standard treatment for many patients with advanced head and neck squamous cell carcinoma (HNSCC). This has led to controversy concerning the role of neck dissection (ND) in this setting. The current debate is focused on N2-N3 disease and the ability of a clinical complete response to predict the absence of viable cells in the ND specimen. Proponents of a systematic planned ND argue that it improves regional control and possibly disease-specific survival. They assert that a clinical response does not predict the pathologic response, and that in the event of recurrence in the neck, a surgical salvage procedure is unlikely to succeed. Conversely, there are many arguments in favor of performing ND only for patients who have evidence of residual neck disease because of the very low probability of isolated neck recurrence following a complete response. Proponents argue that for complete responders, planned ND is associated with no survival benefit. As planned surgery will only benefit patients with residual disease in the neck alone, there is a high rate of unnecessary ND with its associated morbidity. Another question concerns the appropriate type of ND to be performed. Even if required after chemoradiation, selective ND is oncologically feasible with minimal morbidity. Lastly, robust data from a randomized trial demonstrating the superiority of one approach vs. the other are lacking. After conducting a review of recent literature on the subject, the authors conclude that planned ND is not necessary for patients with complete response because of the availability of improved diagnostic follow up modalities, and the increased sensitivity to CRT of HNSCC, particularly HPV associated tumors.Oral Oncology 11/2011; 48(3):203-10. · 2.70 Impact Factor
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ABSTRACT: Since the 1990s we have known of the fascinating ability of a complex set of professional antigen presenting cells (APCs; dendritic cells, monocytes/macrophages, and B lymphocytes) to mediate HIV-1 trans infection of CD4(+) T cells. This results in a burst of virus replication in the T cells that is much greater than that resulting from direct, cis infection of either APC or T cells, or trans infection between T cells. Such APC-to-T cell trans infection first involves a complex set of virus subtype, attachment, entry, and replication patterns that have many similarities among APC, as well as distinct differences related to virus receptors, intracellular trafficking, and productive and nonproductive replication pathways. The end result is that HIV-1 can sequester within the APC for several days and be transmitted via membrane extensions intracellularly and extracellularly to T cells across the virologic synapse. Virus replication requires activated T cells that can develop concurrently with the events of virus transmission. Further research is essential to fill the many gaps in our understanding of these trans infection processes and their role in natural HIV-1 infection.Scientifica. 01/2013; 2013:164203.
JOURNAL OF VIROLOGY, May 2010, p. 4341–4351
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 9
SDF-1/CXCL12 Production by Mature Dendritic Cells Inhibits the
Propagation of X4-Tropic HIV-1 Isolates at the Dendritic
Cell–T-Cell Infectious Synapse?
Nuria Gonza ´lez,1Mercedes Bermejo,1Esther Calonge,1Clare Jolly,2Fernando Arenzana-Seisdedos,3
Jose ´ L. Pablos,4Quentin J. Sattentau,2and Jose ´ Alcamí1*
AIDS Immunopathology Unit, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain1;
The Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom2; Unite ´ de Pathoge ´nie Virale Mole ´culaire,
Institut Pasteur, Paris, France3; and Servicio de Reumatología, Centro de Investigacio ´n, Hospital 12 de Octubre, Madrid, Spain4
Received 20 November 2009/Accepted 15 February 2010
An efficient mode of HIV-1 infection of CD4 lymphocytes occurs in the context of infectious synapses,
where dendritic cells (DCs) enhance HIV-1 transmission to lymphocytes. Emergence of CXCR4-using (X4)
HIV-1 strains occurs late in the course of HIV-1 infection, suggesting that a selective pressure suppresses
the switch from CCR5 (R5) to X4 tropism. We postulated that SDF-1/CXCL12 chemokine production by
DCs could be involved in this process. We observed CXCL12 expression by DCs in vivo in the parafollicular
compartment of lymph nodes. The role of mature monocyte-derived dendritic cells (mMDDCs) in trans-
mitting R5 and X4 HIV-1 strains to autologous lymphocytes was studied using an in vitro infection system.
Using this model, we observed a strong enhancement of lymphocyte infection with R5, but not with X4,
viruses. This lack of DC-mediated enhancement in the propagation of X4 viruses was proportional to
CXCL12 production by mMDDCs. When CXCL12 activity was inhibited with specific neutralizing anti-
bodies or small interfering RNAs (siRNAs), the block to mMDDC transfer of X4 viruses to lymphocytes
was removed. These results suggest that CXCL12 production by DCs resident in lymph nodes represents
an antiviral mechanism in the context of the infectious synapse that could account for the delayed
appearance of X4 viruses.
HIV-1 strains that use CCR5 for entry (R5 strains) are
responsible for most transmission events and predominate in
both early and chronic phases of infection (36, 37), while later
stages of disease are characterized by the frequent emergence
of variants that use both CCR5 and CXCR4 (R5X4 dual-tropic
strains) or CXCR4 alone (X4 strains). About half of the indi-
viduals infected with B clade HIV-1 switch coreceptor use
from CCR5 to CXCR4, and the emergence of X4 viruses is
associated with accelerated CD4?T-cell decline and fast pro-
gression to AIDS (40). The R5-to-X4 switch is associated with
mutations in residues located within the V3 region of gp120,
which tend to increase the overall positive charge of the V3
loop (15). Because only a limited number of mutations are
required for this phenotypic switch (38, 46), the emergence of
X4 variants would be expected to take place on multiple oc-
casions throughout infection. Furthermore, there is evidence
that X4 HIV-1 strains are present as minor viral populations in
patients in whom R5 HIV-1 isolates predominate (11), and the
fast emergence of X4 HIV-1 isolates following treatment with
potent CCR5 antagonists (47) extends that observation. More-
over, CXCR4 expression is more widespread than CCR5 ex-
pression (5, 6). Thus, the failure of X4 HIV-1 to expand during
natural infection is an apparent paradox suggesting the pres-
ence of selective pressures influencing tropism evolution, but
the mechanisms governing such selection are not fully under-
Myeloid and plasmacytoid dendritic cells (PDCs) represent
the two main subsets of DCs that have been described in
humans. Despite sharing common antigens, their functions and
roles in HIV-1 infection are radically different. DCs are the
most potent antigen-presenting cells in vivo (4, 44). Immature
DCs (iDCs) migrate specifically to sites of inflammation to
capture pathogens and pathogen-associated antigens, which
are processed into antigenic peptides and presented on major
histocompatibility complex class II molecules. Once activated
by pathogen encounters, DCs mature and migrate to the T-cell
areas of secondary lymphoid organs, where they interact with
and activate resting T cells and initiate adaptive immune re-
sponses (4, 27). PDCs are located in blood and secondary
lymphoid organs, but they can be recruited to sites of inflam-
mation and are thought to play an important role in innate
immune responses to different types of viruses by producing
alpha interferon (IFN-?).
Certain subsets of DCs residing in the peripheral mucosae
are the first immunocompetent cells to encounter lentiviruses
(21, 39). Successful infection of a host by HIV-1 requires the
dissemination of virus from sites of initial infection at mucosal
surfaces to T-cell zones in secondary lymphoid organs, where
myeloid DCs enhance the infection of CD4?T cells by HIV-1
(10, 33, 34). On the other hand, PDCs inhibit HIV-1 replica-
tion in T cells by secretion of IFN-? and yet-unidentified sol-
uble factors (19). The molecular basis underlying DC–T-cell
spread of HIV-1 remained unclear until the C-type lectin DC-
* Corresponding author. Mailing address: AIDS Immunopathology
Unit, Centro Nacional de Microbiología, Instituto de Salud Carlos III,
Ctra. Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid,
Spain. Phone: (34) 91 8223943. Fax: (34) 91 5097919. E-mail: ppalcami
?Published ahead of print on 24 February 2010.
SIGN (DC specific ICAM-3-grabbing nonintegrin) (18) was
identified. DC-SIGN is highly expressed on DCs present in
mucosal tissues and binds to virus via interaction with the
HIV-1 envelope glycoprotein gp120. DC-SIGN efficiently cap-
tures HIV-1 virions in the periphery and facilitates their trans-
port to secondary lymphoid organs rich in T cells. DCs facili-
tate efficient spread of virus to surrounding permissive T cells
either by infection in trans, in which the DCs present infectious
virus to T cells but are not themselves infected (3, 17, 23), or
in cis, in which the DCs are themselves infected (9, 24).
DCs, macrophages, and intestinal T lymphocytes represent
the primary target cell types during mucosal HIV-1 transmis-
sion. In these cells, CXCR4 is selectively downmodulated (1,
49), because the CXCR4 ligand stromal-cell-derived factor-1
(SDF-1/CXCL12) is constitutively expressed by epithelial cells
within the rectum, endocervix, and vagina (1). Moreover, it has
been reported that intestinal epithelial cells transfer R5 vi-
ruses, but not X4 viruses, to target cells (28). Such mechanisms
may provide a partial explanation for the selection of R5
HIV-1 in the first few days of the infection process during
sexual transmission. However, it is still unclear which processes
drive the predominance of R5 variants early in the course of
parenteral infection and why the emergence of X4 strains,
independently of the route of transmission, occurs late in the
course of HIV-1 infection.
We postulate that CXCL12 production by DCs in lymph
nodes could be involved in these processes. Through this
mechanism, CXCL12 could contribute to the selection of R5
viruses during dissemination in secondary lymphoid organs
after mucosal and intravenous transmission and to the gener-
ally delayed emergence of X4 isolates. To address this hypoth-
esis, we analyzed the impact of CXCL12 on the propagation of
X4 and R5 HIV-1 isolates in an in vitro model of HIV infec-
tion, using an autologous coculture of activated T lymphocytes
and monocyte-derived dendritic cells (MDDCs).
MATERIALS AND METHODS
Ethics. The research performed and management of clinical samples were
approved by the Bioethical Committee of Instituto de Salud Carlos III. When
samples from patients were used to amplify and clone HIV-1 envelopes, in-
formed consent was obtained.
Reagents and cytokines. Interleukin 4 (IL-4) and granulocyte-macrophage
colony-stimulating factor (GM-CSF) were obtained from R&D Systems and
used at 20 ng/ml. Lipopolysaccharide (LPS) from Escherichia coli 055:B5 was
obtained from Sigma and used at 100 ng/ml. Phytohemagglutinin (PHA) and
IL-2 were purchased from Sigma and Chiron, respectively.
Antibodies. The anti-CXCL12 monoclonal antibody (MAb) K15C [IgG2a(?)]
was generated by immunizing BALB/c mice with the CXCL12-derived peptide
KPVSLSYRSPSRFFC conjugated via cysteine 15 to bovine serum albumin
(BSA) (2). K15C and IgG2a (Becton Dickinson) were used at a final concentra-
tion of 30 ?g/ml. The rabbit antisera against HIV-1 Gag p17/p24 were obtained
from the Centers for AIDS Research (CFAR).
For flow cytometry, CD14, CD83, and CXCR4 were detected using phyco-
erythrin-conjugated MAbs from clones M5E2, HB15e, and 12G5 (BD Bio-
sciences), respectively. CCR5 was detected using fluorescein isothiocyanate-
conjugated MAb (clone 2D7; BD Biosciences). For intracellular staining of
CXCL12, cells fixed and permeabilized with 1% paraformaldehyde and 1%
Tween 20 were indirectly stained with the anti-CXCL12 K15C antibody. After
being stained, the cells were analyzed on a FACSCalibur flow cytometer (Becton
Dickinson) using CellQuest software (BD Biosciences).
Cells and cell lines. Peripheral blood mononuclear cells (PBMCs) were ob-
tained from buffy coats of healthy individuals and were purified using Ficoll-
Hypaque density centrifugation (BioWhittaker). Adherent cells were depleted of
lymphocytes by a 1-h plastic adhesion step at 37°C, followed by extensive washing
in prewarmed culture medium. Supernatants containing lymphocytes were col-
lected, and the lymphocytes were grown in RPMI 1640 (BioWhittaker) supple-
mented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin
(100 ?g/ml), and L-glutamine (2 mM) in the presence of IL-2 (300 IU/ml) and
PHA (5 ?g/ml) for 2 days and IL-2 for an additional 5 days. At the time of
coculturing with MDDCs, lymphocytes expressed higher levels of CXCR4 (80 to
90% positive) than of CCR5 (20 to 30% positive). To generate immature
MDDCs (iMDDCs), purified monocytes were cultured in the presence of
IL-4 (20 ng/ml) and GM-CSF (20 ng/ml) for 7 days. At day 5, mature MDDCs
(mMDDCs) were generated by the addition of LPS (100 ng/ml) for 2 days. At
day 7, the phenotype of cultured MDDCs was confirmed by flow cytometric
analysis. mMDDCs expressed high levels of CD83 (70 to 100% positive) and low
levels of CD14 (1 to 5% positive).
293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM)
(BioWhittaker) supplemented with 10% FBS, penicillin (100 U/ml), streptomy-
cin (100 ?g/ml), and L-glutamine (2 mM).
The human T-cell lines Jurkat and MT-2 were grown in RPMI 1640 medium
(BioWhittaker) supplemented with 10% inactivated fetal bovine serum, penicil-
lin (100 U/ml), streptomycin (100 ?g/ml), and L-glutamine (2 mM).
Plasmids. pNL4-3Luc and pNL4-3Ren carry a full-length HIV-1 genome and
the reporter firefly luciferase or Renilla luciferase gene, respectively, cloned into
the nef open reading frame (16). The pJRLuc and pJRRen plasmids were
generated by cloning gp160 from the JR-CSF clone (R5 tropism) in place of the
NL4-3 envelope in the plasmid pNL4-3Luc or pNL4-3Ren (N. Gonza ´lez, M.
Pe ´rez-Olmeda, J. Garcı ´a-Pe ´rez, S. Sa ´nchez-Palomino, E. Mateos, S. Spijkers, A.
Cascajero, A. Alvarez, and J. Alcamı ´, unpublished data). pNP1525Ren was
constructed by replacing the NL4-3 envelope with gp160 from the reference
isolate NP1525 (X4 tropism) in the plasmid pNL4-3Ren. Plasmids carrying env
from HIV-infected patients were generated by replacing the lacZ gene of the
pNL-lacZ/env-Ren vector with gp160 amplified from patients’ plasma. The R5
viruses 11525 and 15214 harbor envelopes from patients with early infection, and the
X4 strains 1116, 5006, and 392 harbor envelopes from HIV-infected patients at an
advanced stage of the disease. The pNL-lacZ/env-Ren vector was generated by
cloning the Renilla luciferase gene in place of nef and replacing the env coding
sequence with the amino-terminal fragment of the lacZ gene (Gonza ´lez et al.,
unpublished). pmaxGFP (Amaxa) encodes the green fluorescent protein (GFP)
from the copepod Pontellina plumata. The psPAX2 HIV packaging construct in-
cludes the HIV gag and pol genes (12). pTRIP-Sym-?U3-MC1 is an HIV-based
lentiviral vector and was provided by P. Charneau (Institute Pasteur, Paris, France).
pcDNA3-VSVG encodes the (VSVG) vesicular stomatitis virus G protein.
Vector construction and siRNAs. The pSUPER construct was described pre-
viously (7). Oligonucleotides expressing small interfering RNA (siRNA) oligo-
nucleotides directed against human CXCL12 mRNA (Table 1) were designed
according to the method of Elbashir et al. (13) and purchased from Sigma. These
oligonucleotides were annealed and ligated into pSUPER downstream of the H1
promoter, giving rise to pSUPER-siCXCL12 constructs.
The pTRIP-siCXCL12 constructs were generated by digesting the H1-siRNA
cassette from pSUPER-siCXCL12 with XbaI and SalI and inserting it into the
NheI and SalI sites in the plasmid pTRIP-Sym-?U3-MC1.
mMDDC transfection with pSUPER-siCXCL12 constructs. mMDDCs (5 ?
106) were transfected with 5 ?g of pSUPER control plasmid or pSUPER-
siCXCL12 constructs with a human dendritic cell nucleofector kit (Amaxa)
according to the manufacturer’s protocol. To test the efficiency of the transfec-
tion, mMDDCs were also transfected with 5 ?g of the control plasmid,
pmaxGFP, and 24 h later, flow cytometric analysis of GFP expression was
Real-time quantitative RT-PCR. Total RNA from MT-2 cells infected with
lentiviruses was extracted with an Rneasy Mini Kit (Qiagen), and contaminating
genomic DNA was removed by incubation with an RNase-Free DNase Set
(Qiagen). Total RNA (1 ?g) was used for first-strand cDNA synthesis with
TABLE 1. Human CXCL12 cDNA target sequences for siRNAsa
siRNAPositionSequence (5? to 3?)
aThree different siRNAs directed against human CXCL12 were designed to
correspond to distinct parts of the CXCL12 mRNA sequence. Target sequences
of the CXCL12 cDNA are shown.
4342GONZA ´LEZ ET AL.J. VIROL.
Im-Prom RT (Promega) using a deoxyribosylthymine (dT) primer. Quantitative
PCR (qPCR) analysis was performed using SYBR green PCR Master Mix
(Applied Biosystem) according to the manufacturer’s recommendations in an
ABI Prism 7000 (Applied Biosystems). The fragments were amplified with the
following primer set: CXCL12 (forward, 5?-TCT GAG AGC TCG CTT GAG
TG-3?; reverse, 5?GTG GAT CGC ATC TAT GCA TG-3?) and ?-ACT as a
reference (forward, 5?-ACA CTG TGC CCA TCT ACG AGG GG-3?; reverse,
5?-TGA TGG AGT TGA AGG TAG TTT CGT GGA T-3?). A standard curve
was constructed for each PCR fragment, the reference, and the target. Ampli-
fication was monitored in real time and allowed to proceed in the exponential
phase until the fluorescent signal reached a significant value (threshold cycle
The formula used for relative quantification was 2???Ct, i.e., the differential
expression of a specific gene between samples.
Generation of virus stocks. 293T cells were transfected with calcium phos-
phate with 10 ?g of the plasmids, and 5 ? 105cells were plated in 6-well tissue
culture plates. The culture medium was replaced with fresh DMEM 8 h and 24 h
after transfection. The cell supernatants were harvested 48 h after transfection
and frozen in aliquots at ?80°C. The amounts of p24 viral antigen in the
supernatants were quantified using commercially available antigen capture en-
zyme-linked immunosorbent assay (ELISA) kits (Innotest HIV antigen MAb;
The transfecting DNA mixture for generating env-pseudotyped lentivirus par-
ticles was composed of 10 ?g of psPAX2, 20 ?g of pTRIP-siCXCL12, and 5 ?g
of the glycoprotein-encoding plasmid pcDNA3-VSVG.
HIV-1 infection assays. MDDCs (3 ? 106to 5 ? 106per well in a 6-well plate)
were incubated with HIV-1 (200 ng of CA-p24) for 2 h at 37°C to allow adsorp-
tion of the virus. The cells were then washed in phosphate-buffered saline (PBS)
to remove unbound virus (the last wash was negative for p24) and cocultured
with activated lymphocytes (5 ? 106) in a 6-well plate. MDDCs or activated
lymphocytes cultured alone were pulsed with the same amount of HIV-1 (200 ng
When the infection assays were performed by adding both viruses, JR-CSF and
NL4-3, in the same well, MDDCs were incubated for 2 h with identical concen-
trations of CA-p24 (200 ng of each). Luciferase activity was measured in cell
lysates 48 h postinfection. Half of the cells in one well were measured using the
Renilla Luciferase Assay System kit and the other half with a firefly luciferase
substrate (Luciferase Assay System).
To study the roles of cis and trans mechanisms of HIV-1 transmission, Trans-
well cell culture plates with 0.4-?m-pore-size polycarbonate membrane inserts
(Corning) were used to separate MDDCs from lymphocytes.
In the neutralization assays with antibodies against CXCL12, MDDCs were
incubated with 30 ?g/ml K15C MAb or an equal amount of isotype-matched
mouse IgG2a for 30 min at 37°C before being cocultured with activated lympho-
cytes. In the assays using siRNA expression constructs, MDDCs were transfected
as described above, and after 30 min, NL4-3Ren or JRRen viruses were added.
In all the infection assays, cells were collected at 48 h for measuring luciferase
activity (relative luciferase units [RLU]) in the cell lysates with a luciferase
reporter assay kit using a Sirius luminometer (Berthold Detection Systems).
Immunohistochemical analysis. Staining of the tissue cryosections was per-
formed as described previously (30). Antigen retrieval was performed by heating
before immunostaining. Endogenous peroxidase was quenched in 3% H2O2in
methanol for 20 min. Staining was performed following a standard indirect
avidin-biotin horseradish peroxidase method and developing with diaminoben-
zidine (Vector Laboratories). Sections were counterstained with hematoxylin.
Immunostaining and confocal microscopy. Cells (MDDCs and Jurkat cells)
were washed with ice-cold PBS, followed by fixation in 2% paraformaldehyde
and 0.025% glutaraldehyde for 10 min at room temperature. The cells were
permeabilized with 0.25% Triton X-100, followed by being blocked with 0.2%
BSA for 10 min at room temperature. Slides were incubated with anti-CXCL12
(K15C) or an isotype-matched control (mouse IgG2a) overnight at 4°C. After
being washed in PBS containing 0.2% BSA, samples were incubated with Alexa
488 goat anti-mouse antibody (Molecular Probes) at 1:500 for 1 h at room
CD4?T cells negatively enriched from PBMCs by magnetic cell sorting
(Miltenyi Biotec) were labeled with the cytoplasmic dye CellTracker green (Mo-
To facilitate conjugate formation, 5 ? 105effector cells (HIV-1-pulsed
mMDDCs) were mixed with an equal number of target cells (activated CD4?T
cells) at 37°C on poly-L-lysine (Sigma-Aldrich)-treated coverslips for 30 min, with
or without the inclusion of MAb K15C. The cells were fixed in 4% formaldehyde
in PBS-1% BSA for 15 min at 4°C. For intracellular staining of HIV-1 Gag, the
conjugates were permeabilized in 0.1% Triton X-100–5% fetal calf serum (FCS).
Rabbit antisera against HIV-1 Gag p17 and p24 were obtained from the CFAR.
Primary antibodies were visualized by single staining using tetramethyl rhoda-
mine isothiocyanate (TRITC)-anti-rabbit IgG (Jackson ImmunoResearch Lab-
oratories). The appropriate controls without primary antibody were performed.
Stained coverslips were analyzed using a Bio-Rad confocal microscope and
processed using Metamorph v6 (Universal Imaging Corporation) and Photoshop
7.0 (Adobe Inc.).
CXCL12 production in lymph nodes and cultures of
MDDCs. We hypothesized that the inefficient transmission
and propagation of X4 HIV strains during early and chronic
infection might be due to CXCL12 expression within second-
ary lymphoid tissue. We therefore analyzed CXCL12 expres-
sion within lymph node tissue and determined the cell types
producing the chemokine by immunohistochemical analysis
using the monoclonal anti-CXCL12 antibody (K15C). As
shown in Fig. 1Ai, CXCL12 staining was observed in endothe-
lial cells, in histiocyte-like cells localized in follicular regions,
and in parafollicular dendritic cells. Using immunofluorescence
techniques (Fig. 1Aii), strong expression of CXCL12 was de-
tected in the paracortical areas of lymph nodes. Interestingly, the
large majority of DC-SIGN-positive cells produced CXCL12,
demonstrating the expression of this chemokine by DCs.
CXCL12 production in an in vitro culture system of MDDCs
was assessed by flow cytometry (Fig. 1Bi) and immunofluores-
cence (Fig. 1Bii). We observed CXCL12 expression in both
iMDDCs and mMDDCs, but production was always higher in
mature than in immature MDDCs. Also, CXCL12 levels in
culture supernatants from MDDCs were quantified by ELISA
at 16 pg/ml in iMDDC and 133 pg/ml in mMDDC cultures.
Enhancement of HIV-1 transfer from mMDDCs to target
cells is higher for R5 viruses. To analyze the role of DCs in the
propagation of R5 and X4 strains, HIV-1-pulsed mMDDCs
were cultured with autologous T cells. mMDDCs were pulsed
for 2 h with R5 or X4 HIV-1 stocks carrying a luciferase
reporter gene using equivalent amounts (200 ng) of Gag p24,
washed, and cultured in the presence of activated T cells. The
cells were assayed for luciferase activity 48 h later (Fig. 2A). As
controls, activated T cells and mMDDCs were challenged sep-
arately with virus using equivalent amounts (200 ng) of Gag
p24. Infection with an R5 HIV-1 strain (JRRen) was increased
by a mean of 7-fold when lymphocytes were cocultured with
autologous dendritic cells compared to direct infection of ac-
tivated lymphocytes with cell-free virus. In contrast, when an
X4 HIV-1 strain (NL4-3Ren) was used, no enhancement in
T-cell infection by dendritic cell-mediated transfer was ob-
served (0.89-fold compared to cell-free virus infection). No
luciferase activity was detected in mMDDCs cultured in the
absence of lymphocytes, suggesting that mMDDCs did not
become infected under these conditions. Conjugates between
mMDDCs pulsed with NL4-3Ren or JRRen and CD4?T cells
were immunostained with MAbs to Gag and analyzed by laser
scanning confocal microscopy (Fig. 2B). Despite detection
of similar levels of Gag protein in mMDDCs, Gag labeling
in lymphocytes was preferentially observed for R5 viruses
(JRRen), while for X4 viruses (NL4-3Ren), Gag protein was
To analyze the enhancement of R5-tropic viruses in the
context of mixed infection with both R5 and X4 viruses and to
VOL. 84, 2010 CXCL12 PRODUCTION BY DCs INHIBITS X4 PROPAGATION4343
simulate conditions where an individual is exposed to both
viruses simultaneously, cells were infected with a mixture of R5
and X4 viruses harboring two different luciferase markers
(Renilla or firefly). Luciferase activity was also measured in cul-
tures infected singly (monoinfection) with each virus using
equivalent concentrations of Gag p24. As shown in Fig. 3,
the outcomes were very similar when lymphocytes were in-
fected with single or mixed viral preparations. There was a
preferential transmission of R5 over X4 viruses when both
were present at similar concentrations in T-cell–mMDDC
To confirm these results with recombinant viruses carry-
ing envelope glycoproteins from clinical viral isolates,
mMDDCs were incubated with recombinant viruses encod-
ing R5 and X4 envelopes amplified directly from patient
viral populations (Fig. 4). The profile shown by the infection
with recombinant viruses generated from HIV-1-infected
patients was very similar to the one presented by reference
R5 and X4 viruses.
X4 HIV-1 transmission from mMDDCs to lymphocytes is
lower than from iMDDCs. As higher levels of CXCL12 produc-
tion were detected in mMDDCs than in iMDDCs (Fig. 1B), we
analyzed the enhancement of viral propagation using iMDDCs or
mMDDCs pulsed with R5 or X4 viruses (Fig. 5A). In contrast
with the absence of infection enhancement observed when lym-
phocytes were cultured with X4-pulsed mMDDCs, enhancement
was observed when lymphocytes were cocultured with X4-pulsed
iMDDCs. These data correlate with the levels of CXCL12 pro-
duction and suggest that CXCL12 produced by mMDDCs inter-
feres with the transmission of X4-tropic strains. As expected,
similar levels of enhancement in R5 infection were detected when
lymphocytes were cocultured with either iMDDCs or mMDDCs.
FIG. 1. CXCL12 expression. (A) CXCL12 immunostaining in lymph nodes. (i) Cryosections from normal lymph nodes were examined for the
expression of CXCL12 using the K15C specific monoclonal antibody. In the parafollicular zone, there were cells with dendritic morphology positive
for CXCL12 staining (left; original magnification, ?100). In germinal centers, cells displaying a histiocyte-like morphology showed CXCL12
immunostaining (middle; original magnification, ?100). Endothelial cells were also immunostained by anti-CXCL12 MAb (arrows in right panel;
original magnification, ?20). Sections were counterstained with hematoxylin. (ii) Staining with CXCL12 (left; green) and DC-SIGN (center; red)
in lymph nodes using immunofluorescence techniques showed double labeling of DCs (right; merged). (B) Increased CXCL12 expression in
mature dendritic cells. (i) Flow cytometry. Cell surface expression of the maturation marker CD83 was determined by direct immunofluorescence
and intracellular expression of CXCL12 by indirect immunofluorescence. The percentages of positive cells are shown in the graph. The
fluorescence values of appropriate isotype controls are represented by gray filled lines. (ii) CXCL12 expression in iMDDCs and mMDDCs by
confocal microscopy. iMDDCs and mMDDCs were plated on poly-L-lysine-coated coverslips, fixed with formaldehyde, permeabilized, and stained
4344GONZA ´LEZ ET AL. J. VIROL.
HIV-1 transmission from MDDCs to lymphocytes does not
occur in Transwell cultures. To get a better insight into the
roles of cis and trans mechanisms of HIV transmission from
dendritic cells to lymphocytes, we performed Transwell assays
in which iMDDCs or mMDDCs were seeded in lower wells
and T cells were seeded in upper wells for infection assays with
X4 and R5 viruses. As shown in Fig. 5B, in the absence of
cell-to-cell contact, no infection was detected in T cells seeded
in the upper wells. This finding strongly suggests that under the
conditions tested, T lymphocytes were infected in trans through
transfer from noninfected DCs and not in cis by virions re-
leased from infected dendritic cells.
CXCL12 neutralization enhances X4 HIV-1 infection of lym-
phocytes cultured with autologous mMDDCs. HIV-1-pulsed
mMDDCs were preincubated with antibodies against CXCL12
prior to the addition of lymphocytes. The use of the CXCL12
neutralizing antibody K15C increased infection with X4 viruses
2.4-fold (Fig. 6A). These data indicate that infection with an
X4 HIV-1 strain was inhibited when lymphocytes were cocul-
tured with mMDDCs and that this inhibition was dependent
on CXCL12 production. In contrast, infection with R5 HIV-1
strains was not modified in this coculture model, and anti-
CXCL12 antibody had no effect on infection with R5 viruses.
Finally, we analyzed the effect of CXCL12 on HIV-1 transfer
FIG. 2. mMDDCs enhance R5, but not X4, HIV infection. (A) mMDDCs were incubated with NL4-3Ren (NL) (X4 HIV-1) or JRRen (JR)
(R5 HIV-1). Activated lymphocytes were infected with NL4-3Ren or JRRen. mMDDCs were pulsed with NL4-3Ren or JRRen, washed with PBS
to remove unadsorbed virus, and cocultured with activated lymphocytes. RLU were determined 48 h after infection. Shown are the means and
standard errors of the mean (SEM) of 15 experiments. (B) Conjugates between NL4-3Ren- or JRRen-pulsed mMDDCs and CD4?T cells were
fixed, permeabilized, and stained by indirect immunofluorescence with a MAb specific for Gag (red). CD4?T cells were prelabeled with a
cytoplasmic dye (green). A three-dimensional reconstruction of a z series of images is shown. One representative field out of 20 is shown.
VOL. 84, 2010 CXCL12 PRODUCTION BY DCs INHIBITS X4 PROPAGATION4345
by laser scanning confocal microscopy. For this, we established
a system based on the formation of conjugates between
mMDDCs pulsed with NL4-3Ren virus and CD4?T cells. Such
conjugates were formed at 37°C in the presence or absence of
the antibody K15C, and conjugates were examined for viral
Gag localization. When the antibody against CXCL12 was
added, HIV-1 was often detected in the conjugated T cells,
showing transfer of X4 virus to lymphocytes (Fig. 6B). On the
other hand, the Gag protein detection levels in T cells did not
increase for R5 viruses when the antibody K15C was added
CXCL12 siRNAs enhance X4 HIV-1 infection of lymphocytes
cultured with autologous mMDDCs. With the aim of silencing
human CXCL12 expression, we designed siRNAs targeting the
CXCL12 gene (Table 1). The capacity of siRNAs to inhibit
CXCL12 expression was tested by cotransfection of Jurkat
cells with a control plasmid (pSUPER-siGFP) or pSUPER-
siCXCL12 constructs (pSUPER-siCXCL12 94, pSUPER-
siCXCL12 183, and pSUPER-siCXCL12 244), together with a
human CXCL12 expression plasmid. Cells were stained with
the anti-CXCL12 K15C MAb, and protein expression was an-
alyzed by immunofluorescence (Fig. 7A). Furthermore, siRNA
FIG. 3. Coinfection with R5 and X4 viruses. Dual-infection assays
involved the addition of the two HIV-1 isolates (JR and NL) at similar
concentrations carrying different luciferase reporters, firefly and Re-
nilla, and were performed alongside the monoinfections. The graph
shows the RLU values in monoinfected cells and in dual-infection
assays using an R5 and an X4 virus with different luciferase markers.
On the left, measurement of luciferase reflected infection of NL4-3
(X4-tropic virus) under different conditions (mono- or dual infection).
On the right, the luciferase levels indicated infection with the JR-CSF
strain (R5-tropic virus). Shown are the means and SEM of four ex-
FIG. 4. mMDDCs enhance infection of R5, but not X4, HIV-1
isolates carrying the envelope from patients’ isolates. Lymphocytes
were activated with PHA and IL-2 for 7 days and infected with recom-
binant viruses carrying Env from HIV-infected patients (white bars).
mMDDCs were pulsed with these viruses, washed with PBS to remove
unadsorbed virus, and cocultured with activated lymphocytes (black
bars). RLU were determined 48 h after infection. JR, 11525, and 15214
are R5 viruses. NL, NP1525, 1116, 5006, and 392 are pure X4 viruses.
Shown are the means and SEM of three experiments.
FIG. 5. (A) Increased transmission of X4 strains to lymphocytes is
produced by iMDDCs, but not by mMDDCs. Infection of lymphocytes
cocultured with iMDDCs is represented by gray bars. The black bars
correspond to the infection of lymphocytes cocultured with mMDDCs.
Shown are the means and SEM of three experiments. (B) HIV-1
transmission from MDDCs to lymphocytes does not occur in Transwell
cultures. mMDDCs or iMDDCs were incubated with NL4-3Ren or
JRRen, washed, and cocultured with activated lymphocytes in the
same well (T cells and i/mMDDCs) or separated by a polycarbonate
membrane (Transwell). When the Transwell membrane was present,
cell lysates were obtained from both the upper compartment (T cells)
and the lower well (MDDCs). RLU were determined 48 h after infec-
tion. One experiment out of two is shown.
4346GONZA ´LEZ ET AL.J. VIROL.
knockdown of CXCL12 expression was confirmed by qPCR.
As observed in Fig. 7B, transduction of MT-2 cells, which
express CXCL12 naturally, with CXCL12 siRNA-expressing
lentiviruses provoked a significant decrease in the expression
of the chemokine compared to cells infected with control
siRNA-expressing lentivirus. The ability of siRNAs to inhibit
CXCL12 expression in mMDDCs was confirmed by immuno-
fluorescence (Fig. 7C).
We studied the effects of CXCL12-specific siRNAs on
HIV-1 trans-enhancement of activated T cells. For this pur-
pose, mMDDCs were transfected with the control plasmid
pSUPER-siGFP or with the three pSUPER-siCXCL12 con-
structs (pSUPER-siCXCL12 94, pSUPER-siCXCL12 183,
and pSUPER-siCXCL12 244). The efficiency of the trans-
fection of mMDDCs (25%) was measured by transfecting
these cells with the plasmid pmaxGFP and analysis by flow
cytometry (Fig. 7D). After 30 min, JRRen or NL4-3Ren was
added and incubated for 2 h before the addition of lympho-
cytes. mMDDCs transfected with the mixture of pSUPER-
siCXCL12 vectors were 3-fold more efficient at transferring
NL4-3Ren to activated lymphocytes than mMDDCs trans-
fected with the control vector (Fig. 7D). This increase in in-
FIG. 6. Blocking of the CXCL12 effect by the anti-CXCL12 antibody K15C increased infection of HIV-1 X4 strains. (A) mMDDCs were pulsed for
2 h with NL4-3Ren or JRRen, and unbound virus particles were washed away. Then, the mMDDCs were preincubated with the neutralizing antibody
(Ab) K15C (gray bars) or an isotype control (black bars) for 30 min at 37°C and cocultured with activated lymphocytes. Shown are the averages and SEM
of three experiments. (B) Conjugates were formed between NL4-3Ren- or JRRen-pulsed mMDDCs and CD4?T cells in the absence or presence of
K15C. The cells were fixed, permeabilized, and stained for Gag (red). CD4?T cells were prelabeled with a cytoplasmic dye (green).
VOL. 84, 2010CXCL12 PRODUCTION BY DCs INHIBITS X4 PROPAGATION4347
fection did not occur in mMDDCs transfected with
pSUPER-siCXCL12 constructs and pulsed with the virus JRRen
compared to those transfected with the control plasmid. These
data confirmed that CXCL12 produced by mMDDCs inter-
fered with the transmission of X4 strains.
DCs capture HIV-1 at mucosal entry sites and transport
virus to the T-cell compartment in lymphoid tissues, where
DC-associated HIV-1 is efficiently transmitted to CD4?T cells
(10, 33). HIV-1 specifically binds to DCs through the interac-
tion of gp120 with DC-SIGN (17) or other lectin-like receptors
(42). Once present within lymph nodes, HIV-1 may pass di-
rectly from DCs to T cells in the parafollicular area or may
become trapped on follicular dendritic cells in lymphoid folli-
The observation that transmission of infection is confined to
R5 strains of HIV-1 has remained an enigma. The selective R5
HIV-1 transmission via sexual intercourse could be caused by
FIG. 7. siRNA directed against CXCL12 increased infection by HIV-1 X4 strains. (A) CXCL12 expression in Jurkat cells that were untransfected
or transfected with a plasmid control or with siRNA expression constructs against CXCL12. (B) qPCR analysis of CXCL12 mRNA expression in MT-2
cells that were uninfected (white bars) or infected with control siRNA-expressing lentiviruses (black bars) or with CXCL12 siRNA-expressing lentiviruses
(gray bars). Shown are the means and SEM from two experiments. (C) CXCL12 expression in mMDDCs that were untransfected or transfected with
expression vectors against CXCL12 (gray bars), pulsed with NL4-3Ren or JRRen, and incubated with lymphocytes. The direct infection of lymphocytes
is represented by white bars. One representative experiment out of three is shown. The inset shows the efficiency of transfection of mMDDCs with
4348GONZA ´LEZ ET AL. J. VIROL.
CXCL12 production by cells at mucosal surfaces, which may
reduce the transmission and propagation of X4 HIV-1 at these
sites (1). This reduced transmission of X4 HIV-1 correlates
with the predominantly intracellular expression of CXCR4 in
resident skin Langerhans cells (49) and intestinal intraepithe-
lial lymphocytes (1). CXCL12 protein and mRNA expression
have also been observed in endothelial cells, pericytes, and
CD1a?dendritic cells (30). However, these findings do not
explain why R5 isolates are also selected in primary infection
via the intravenous route or the persistence of R5-tropic
strains for years in HIV-1-infected patients. The latter obser-
vation is particularly difficult to understand, since relatively few
mutations in the highly variable V3 loop can account for a
switch of HIV-1 coreceptor use from CCR5 to CXCR4 (38,
46). Despite the fact that in some particular contexts, such as
treatment with CCR5 antagonists, multiple changes in the re-
ceptor do not have as a consequence a switch to CXCR4 use
(25), in vivo the earliest detectable X4 variants show only one
or two amino acid substitutions compared to coexisting R5
variants (22). These observations point to the existence of
selective pressure against X4 HIV-1 evolution, the exact na-
ture of which remains to be established. A role for humoral
immunity in HIV-1 tropism evolution has been proposed, and
in some patients, the increased neutralization of recently
emerged X4 strains can potentially contribute to the late emer-
gence of X4 variants. However, in other patients, the absence
of neutralizing antibodies against X4 viruses suggests that
other selective pressures are also involved (8).
Based upon the data presented in the current study, and
particularly CXCL12 production by DC-SIGN-positive DCs in
the parafollicular T-cell areas of lymph nodes, we propose that
CXCL12 production at the DC–T-cell infectious synapse (31)
is a selective pressure interfering with X4 HIV-1 use of
CXCR4 and thereby the emergence and propagation of X4
HIV-1 strains in vivo. In further support of this proposal, we
demonstrated high expression levels of CXCL12 by a variety of
cell types, mainly endothelial and stromal cells in the parafol-
licular T-cell area in lymph nodes. CXCL12 production by
these cell types produces a general tissue environment contain-
ing high local CXCL12 levels that would not favor the propa-
gation of X4 viruses.
According to this hypothesis, a decrease in CXCL12 pro-
duction secondary to the destruction of lymph node architec-
ture could contribute, together with other mechanisms of im-
mune failure, to the emergence of viruses using the CXCR4
receptor in advanced stages of HIV disease. As an alternative
mechanism, the switch from R5- to R5X4- or X4-tropic viruses
could be related to the generation of viral envelopes displaying
high avidity for CXCR4 that would compete efficiently with
CXCL12 binding to the receptor.
Using an in vitro infection system of lymphocytes cocultured
with mMDDCs previously pulsed with HIV-1, we observed
that transmission and propagation of X4 viruses was worsened
in comparison with R5 strains. Interestingly, the inhibitory
effect provided by mMDDCs was even stronger for viral chi-
meras carrying the viral envelope from HIV-infected patients
than for the reference laboratory-adapted strain NL4-3. Over-
all, these results confirm the lack of trans-enhancement for X4
viruses in the infectious synapse, in contrast with R5-tropic
strains. This difference between R5 and X4 virus infections was
not due to higher levels of CCR5 expression on target lympho-
cytes, as described by other authors (20). Actually, after treat-
ment with PHA and IL-2, CXCR4 was expressed in 80 to 90%
of lymphocytes, whereas CCR5 expression was detected in 20
to 30% (data not shown). It has been reported that intracel-
lular expression of Nef protein from HIV-1 and other lentivi-
ruses downregulates CXCR4 from the cell membrane (29, 45).
The recombinant viruses used do not express Nef, and there-
fore, the effect is not relevant to this experimental system.
However, it can be argued that in the presence of Nef de-
creased infection of lymphocytes in the infectious synapse
could be related to CXCR4 downregulation secondary to Nef
expression and not to an inhibitory effect of CXCL12. How-
ever, CXCR4 downregulation occurs in infected cells express-
ing Nef, and therefore, at least for the first round of infection,
T lymphocytes would be susceptible to infection with CXCR4
strains. Furthermore, downregulation induced by Nef has been
reported, not only for CXCR4, but also for CCR5 and other
chemokine receptors. The different patterns of R5 and X4
transmission observed in the dendritic cell-lymphocyte envi-
ronment cannot be attributed to this mechanism.
The worst transmission of X4 viruses was related to CXCL12
production. By blocking studies with CXCL12 neutralizing an-
tibody or by siRNA knockdown of CXCL12 expression in DCs,
X4 virus trans-enhancement was mediated in DC–T-cell con-
jugates. In contrast, the trans-enhancement of R5 virus infec-
tion from DCs to T cells was predictably not affected by the
high levels of CXCL12 expression, and this was the case even
when R5 virus was introduced in the presence of an equivalent
amount of X4 virus.
Yamamoto et al. (48) observed selective replication of
R5 over X4 HIV-1 isolates in CD4?T cells cultured with
iMDDCs. This increased infectivity was dependent on the
activation state of CD4?T cells, since weakly activated CD4?
T cells were preferentially infected by R5 viruses. Our results
agree with those of Yamamoto et al., as we observed that in a
coculture of iMDDCs and PHA-stimulated T cells, both R5
and X4 viruses were able to replicate at similar levels. In
contrast, we found preferential replication of R5 viruses in
PHA-activated lymphocytes when they were cocultured with
mMDDCs. Therefore, the activation state of CD4?T cells
would not be the only factor implicated in R5 selection in the
Previous studies have found an enhancement of both X4 and
R5 viruses mediated by DC-SIGN. However, in these experi-
ments, DC-SIGN-expressing immortalized cell lines (17, 41) or
immature dendritic cells (14, 23, 32, 41) were used, which
either did not express CXCL12 or produced only small
amounts of the chemokine. As in previously published studies,
in our in vitro system, when lymphocytes were cocultured with
iMDDCs, trans-enhancement of HIV-1 infection was observed
both when R5 HIV-1 was used and when X4 HIV-1 was used.
Other studies have described increased transmission of X4
HIV-1 by mature dendritic cells (26, 35). Differences in the
stimuli employed for MDDC maturation and the use of cell
lines or purified naïve lymphocytes as target cells could ac-
count for this discrepancy (26, 35). Also, higher (between 10
and 50 times) viral inputs were used in those experiments than
in our experimental model (26, 35), which could potentially
VOL. 84, 2010 CXCL12 PRODUCTION BY DCs INHIBITS X4 PROPAGATION4349
overwhelm the blocking capacity of CXCL12 produced by den-
A two-phase transfer of HIV-1 from MDDCs to lympho-
cytes has been previously described elsewhere as follows: an
early short-term phase of viral transfer in trans, caused by the
direct binding of viral particles to DC-SIGN or other mole-
cules at the surfaces of the MDDCs, and then a later long-term
transfer phase (from 48 h to 72 h after viral exposure) that
depends on the ability of MDDCs to complete de novo viral
production (9, 43). However, for our lymphocyte-MDDC co-
culture model’s HIV-1 exposure of less than 48 h, HIV-1 trans-
fer to lymphocytes secondary to MDDC infections probably
does not take place, which is suggested by the lack of luciferase
activity detection in our pure MDDC cultures. The results
from Transwell experiments strongly suggest that under the
conditions tested, lymphocytes were infected in trans through
transfer from noninfected MDDCs and not in cis by virions
released from productively infected dendritic cells. Neverthe-
less, CXCL12 production by mMDDCs could selectively inter-
fere with the transmission of X4 HIV-1 isolates to neighboring
CD4 T lymphocytes during both the early and later phases
after viral exposure.
In summary, we propose that during the process of matura-
tion of dendritic cells, the expression of the chemokine
CXCL12 is upregulated. As a consequence, increased CXCL12
production by mDCs could naturally and selectively block the
transmission of X4 HIV-1 strains after HIV-1 exposure. This
barrier could partly explain the lack of X4 virus detection
during early and chronic HIV-1 infection.
This work was supported by the following projects: Spanish Ministry of
Research (SAF 2004-04258); Spanish Health Ministry (ISCIII-RETIC
RD06/0006); Instituto de Salud Carlos III (PI05/00017); VIRHOST Net-
work from Comunidad de Madrid, Spain; Fundacio ´n Mutua Madrilen ˜a;
and Agence Nationale de la Recherche sur le SIDA (ANRS), France.
Financial support was also provided through EUROPRISE. N.G. was
supported by a fellowship from the Spanish Ministry of Education and
Science (FPI fellowship SAF00/0028).
We thank Antonio Caruz for advice on siRNA design. We acknowl-
edge Olga Palao for excellent secretarial assistance, and we also thank
The Center of Blood Transfusions (Comunidad de Madrid) for sup-
plying the blood of healthy donors.
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