JOURNAL OF VIROLOGY, May 2005, p. 5386–5399
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 9
Covert Human Immunodeficiency Virus Replication in Dendritic Cells
and in DC-SIGN-Expressing Cells Promotes Long-Term
Transmission to Lymphocytes
Cinzia Nobile,1Caroline Petit,2Arnaud Moris,1Katharina Skrabal,3Jean-Pierre Abastado,4
Fabrizio Mammano,3and Olivier Schwartz1*
Groupe Virus et Immunite ´, URA CNRS 1930, Institut Pasteur,1Institut Cochin,2INSERM U552-Recherche Antivirale,
Ho ˆpital Bichat,3and IDM Research Laboratories, Institut des Cordeliers,4Paris, France
Received 27 September 2004/Accepted 6 December 2004
HIV-1 virions are efficiently captured by monocyte-derived immature dendritic cells (iDCs), as well as by cell
lines expressing the lectin DC-SIGN. Viral infectivity can be retained for several days, and even enhanced,
before transmission to CD4?lymphocytes. The role of DC-SIGN in viral retention and enhancement of
infection is not fully understood and varies according to the cell line expressing the lectin. We studied here the
mechanisms underlying this process. We focused our study on X4-tropic human immunodeficiency virus (HIV)
strains, since they were widely believed not to replicate in iDCs. However, we first show that X4 HIV replicates
covertly and slowly in iDCs. This is also the case in Raji–DC-SIGN cells, which are classically used to study
HIV transmission. We used either single-cycle or replicative HIV and measured viral RT and replication to
further demonstrate that transfer of incoming virions from iDCs or DC-SIGN?cells occurs only on the
short-term (i.e., a few hours after viral exposure). There is no long-term storage of original HIV particles in
these cells. A few days after viral exposure, replicative viruses, and not single-cycle virions, are transmitted to
CD4?cells. The cell-type-dependent activity of DC-SIGN reflects the ability of HIV to replicate covertly in some
cells, and not in others.
Human immunodeficiency virus (HIV) subverts the traffick-
ing properties of dendritic cells (DCs) to reach secondary lym-
phoid organs and to spread to CD4?T lymphocytes. Virions
are steadily captured by DCs. Conjugates between DCs and T
cells are easily formed (46, 50), a process which facilitates
transmission of HIV by locally concentrating virus on donor
cells and viral receptors on target cells during the formation of
an infectious synapse (31, 41). In DCs, a variety of molecules
can bind gp120, the viral envelope glycoprotein (19, 27, 55, 56).
Among them, DC-SIGN (or CD209), a C-type (Ca-dependent)
lectin that selectively recognizes high-mannose oligosaccha-
rides (17), plays a peculiar role during virus transmission. DC-
SIGN is expressed on some DC subsets, including those de-
rived from blood monocytes or found in lymphoid tissues and
beneath genital surfaces (30). DC-SIGN-expressing cells inter-
nalize HIV type 1 (HIV-1) virions into a trypsin-resistant com-
partment, and viral infectivity can be retained for several days
and even enhanced before transmission to T cells (2, 6, 19, 36,
54). In addition to DC-SIGN, the CD4 molecule and other
lectins such as the mannose receptor and langerin bind gp120
(56), and they may also play a role in virus capture and trans-
The mechanisms by which DC-SIGN promotes trans infec-
tion of target cells are not fully understood. Incoming virions
are rapidly degraded in DCs and in DC-SIGN?cells (43, 57).
There is thus an apparent discrepancy between the short half-
life of incoming virions (?3 h) and the ability of DCs and
DC-SIGN?cell lines to retain and transmit the infection to T
cells, which has been observed up to 6 days after viral exposure
(19, 45, 54).
Several mechanisms may account for this discordance. First,
defective virions, which form the majority of viral preparations,
may be more sensitive to degradation than fully infectious
particles. Second, cell-cell transmission of virus through the
infectious synapse is an efficient and rapid process that may
necessitate only minute amounts of virions (26, 41). DC-SIGN
internalizes HIV into a low-pH compartment (36). It has been
proposed that virus recycling from this compartment will lead
to infectivity enhancement and transmission (36). This intra-
cellular compartment has not yet been identified, and the na-
ture of the pH-dependent process enhancing infectivity is not
understood. Interestingly, the properties of DC-SIGN when
expressed on immature DCs (iDCs) can be recapitulated in
Raji DC-SIGN B cells, which allow viral transfer on the long
term, as well as DC-SIGN-mediated trans enhancement (19,
36, 54, 58). However, a cell-type-dependent activity of DC-
SIGN has been documented, and other cell lines, such as
THP1, K562, or 293 cells expressing the lectin, are unable to
perform these tasks (54, 58, 59). The origin of this cell-type-
dependent effect has not been deciphered.
Third, a nonexclusive possibility is that progeny viruses,
rather than input virions, are transmitted from DCs to lym-
phocytes. HIV-1 replicates rather inefficiently in DC cultures.
DCs express low levels of CD4 and coreceptors CCR5 or
CXCR4. It has been reported that R5, but not X4, HIV-1
strains replicate in immature monocyte-derived DCs (21, 45,
47). However, both R5 and X4 viruses readily enter DCs and
are able to perform reverse transcription (RT) in these cells (3,
9, 14, 22–24). Of note, both X4 and R5 strains are efficiently
* Corresponding author. Mailing address: Institut Pasteur, Groupe
Virus et Immunite ´, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France.
Phone: 33 1 45 68 83 53. Fax: 33 1 40 61 34 65. E-mail: schwartz
transmitted, even a few days after viral exposure, from DCs to
lymphocytes (19, 45). However, with R5 strains, which repli-
cate in DCs, viral progeny (but not incoming virions) are trans-
mitted after a few hours (57). It is thus conceivable that a
previously underestimated, surreptitious replication of X4 iso-
lates in DCs will be the main source of an infection spreading
towards the lymphocytes.
In this report, we examined the process of HIV transmission
from iDCs or DC-SIGN-expressing cells to target cells. We
focused our study on X4 strains, since they were previously
believed not to replicate in iDCs. In contrast to these initial
reports, we show that X4 strains replicate consistently at low
levels in monocyte-derived iDCs. By using either single-cycle
or replicative HIV and measuring viral RT and replication, we
further demonstrate that a short-term (i.e., a few hours after
viral exposure), pH-independent transfer of incoming virions
occurs in iDCs or in DC-SIGN-expressing cells. However,
there is no long-term storage of infectious HIV particles in
these cells. A few days after viral exposure, progeny viruses
(rather than incoming virions) are transmitted to lymphocytes.
The cell-type-dependent activity of DC-SIGN on long-term
virus transmission simply reflects the ability of HIV to replicate
covertly in some cells and not in others.
MATERIALS AND METHODS
Cells and reagents. DCs were prepared with a VacCell processor as previously
described (20). Briefly, peripheral blood mononuclear cells from leukapheresis
were cultured for 7 days in serum-free VacCell medium (Invitrogen) supple-
mented with 500 U of granulocyte-macrophage colony-stimulating factor (No-
vartis, France)/ml and 50 ng of interleukin-13 (Sanofi-Synthelabo, France)/ml,
and DCs were isolated by elutriation. The DC isolation procedure yielded
CD1a?, major histocompatibility complex class I- and class II-, DC-SIGN?,
CD64?, CD83?, CD80-low, and CD86-low cells, a phenotype corresponding to
iDCs. Preparations routinely contained about 2% CD3?cells. C1RA2 DC-SIGN
cells were derived from C1R-A2 by infection with a lentiviral vector encoding
DC-SIGN and sorting DC-SIGN-positive cells (43). Raji and Raji DC-SIGN
cells were a kind gift of Ali Amara (19, 58). P4 cells are HeLa CD4?cells
carrying a HIV long terminal repeat (LTR) lacZ reporter cassette. HeLa cells
were transiently transfected with plasmids encoding CD4 and/or DC-SIGN as
previously described (37, 51). Cells were infected with HIV 24 h after transfec-
tion. Bafilomycin A1 and concanamycin A were from Sigma, T-20 was from
American peptide, and zidovudine (AZT) and nevirapine (NVP) were from the
National Institutes of Health AIDS Research and Reference Reagent Program.
Viruses and infections. HIV (NL4.3 and NLAD8 strains) and HIV(vesicular
stomatitis virus [VSV]) virions were produced and titrated as previously de-
scribed (40). NLAD8 was a kind gift of Eric Freed. NL F522Y provirus encodes
a nonfusogenic gp120/g41 complex (8, 11). Single-cycle pseudotyped HIV parti-
cles [HIV-Luc and HIV-Luc(VSV)] were generated by cotransfection of 293T
cells with the proviral vector pNL-Luc E-R- containing the firefly luciferase gene
(15) and with an gp120/gp41 expression vector (from the X4 strain HXB2) or the
VSV-G envelope glycoproteins, respectively. Proviral vectors encoding for pa-
tient-derived envelope glycoproteins were generated by cloning primary env
sequences, spanning the entire gp120 domain and most of gp41, into a pNL4-3
variant (pNL43XC-MS2), which harbors an MluI site in C1 of env (nucleotide
6435) in addition to the natural BamHI site (nucleotide 8465). The primary
envelope genes were obtained by RT-PCR amplification from a plasma sample
issued from a treatment-naive, symptomatic patient and were cloned in MluI-
BamHI. Several replication competent viral clones were obtained and sequenced
(K. Skrabal et al., unpublished data). The tropism of the corresponding viruses
was determined by infection of U373-MG-CD4-CCR5 and U373-MG-CD4-
CXCR4 reporter cells. Infection of iDCs and other cells was described previously
(45). iDCs (106) were exposed to the indicated viruses for 2 to 3 h at 37°C,
extensively washed, and grown for the indicated periods of time. Virus release
was monitored by measuring p24 production in supernatants by enzyme-linked
immunosorbent assay (ELISA) (Perkin-Elmer Life Sciences). When stated,
AZT, NVP, or T-20 was added 1 h before virus exposure and maintained
throughout the assay.
Flow cytometry. Cells were stained with anti-DC-SIGN (monoclonal antibody
[MAb] 161-PE; R&D), anti-mannose receptor (MR) (MAb 3.29B1.10; Coulter),
anti-CD3 (SK7-PE; Becton Dickinson) or isotype MAbs as a negative control;
permeabilized; and intracellularly stained with anti-Gagp24-FITC MAb (KC57;
Coulter). Cells were analyzed by flow cytometry with a FACSCalibur cytometer
HIV capture assay. iDCs were exposed to the indicated viruses (30 or 6 ng of
p24/5 per 105cells) for 2 h at 37°C. Cells were then extensively washed and lysed
in 0.5% Triton buffer. Lysates were centrifuged (5 min at 10,000 rpm) to discard
cell debris before p24 measurement by ELISA.
HIV transmission assay. Donor cells (5 ? 105to 1.5 ? 106) were incubated
with the indicated doses of HIV for 2 h at 37°C. Cells were then washed
thoroughly and 2 ? 104donor cells were added to an equal number of target cells
(HeLa, HeLa CD4?, MT4, or Jurkat cells), either immediately or after the
indicated periods of time. For experiments performed with single-cycle HIV-Luc,
cells were harvested after an additional 48-h culture period. Luciferase activity
was measured with a luciferase reporter assay kit (Promega) and a luminometer
(Perkin-Elmer). Of note, in some experiments (see Fig. 5 and 8), luciferase
activities were measured with another luminometer (Berthold Technologies),
which yielded higher luminescence signals. Luciferase levels cannot thus be
compared to values depicted in some figures (e.g., see Fig. 4, 6, and 9). For
experiments performed with replicative HIV, virus transmission was assessed by
measuring ?-galactosidase activity in P4 cell extracts after a 2-day coculture
period. When stated, bafilomycin A1 or concanamicyn A were added 1 h before
and maintained during virus exposure. Cells were then extensively washed to
remove drugs and grown in culture medium.
Quantitative PCR. To minimize contamination of HIV stocks with plasmidic
or cellular DNA, NL4.3 and NLAD8 viruses were produced by infection of
MT4-CCR5 cells and harvesting cell supernatants before the occurrence of
virus-induced cytopathic effects. NL F522Y (and wild-type [WT] NL4.3 as a
control) virus stocks were prepared by a two-step procedure. First, VSV-G-
pseudotyped virions were generated by cotransfection of HeLa cells. Second, the
resulting viral supernatants were used to infect Jurkat cells. Supernatants of
Jurkat cells were collected a few days after infection and used for further studies.
HIV infections were performed with the indicated viruses (at 50 ng of p24/ml/106
cells). At various time points, DNA was prepared from infected cells with the
Qiaamp DNA extraction kit (Qiagen). Total HIV DNA was measured with
primers in the RU5 LTR region, as previously described (10). Sequences of the
primers (purchased from TIB MOLBIOL) were as follows: forward primer M667
(5?-GGC TAA CTA GGG AAC CCA CTG-3?) and reverse primer AASM
(5?-GCT AGA GAT TTT CCA CAC TGA CTAA-3?). The fluorogenic hybrid-
ization probes used to detect the amplification products were LTR-FL (5?-CAC
AAC AGA CGG GCA CAC ACT ACT TGA-3?) and LTR-LC (5?-LC Red640-
5?-CAC TCA AGG CAA GCT TTA TTG AGG C p-3?). After initial incubation
at 95°C for 8 min, 50 cycles of amplification were carried out for 10 s at 95°C,
followed by 10 s at 60°C and 8 s at 72°C. Complete RT products were quantified
with primers in the Pol region (HIV-Pol F forward, 5?-TTT AGA TGG AAT
AGA TAA GGC CCA A-3?; HIV-Pol R reverse, 5?-CAG CTG GCT ACT ATT
TCT TTT GCT A-3?); the fluorogenic TaqMan probe used to detect the ampli-
fication products was Pol_TM (5?- 6-carboxyfluorescein-AAT CAC TAG CCA
TTG CTC TCC AAT TAC). After initial incubation at 95°C for 8 min, 50 cycles
of amplification were carried out, each consisting of 5 s at 95°C, followed by 30 s
at 62°C. Reaction mixtures contained 1? LightCycler-FastStart DNA Master
Hybridization probes (Roche), 300 nM each primer, 200 nM each probe, and 5
?l of template DNA in a 20-?l volume. For each experiment, a standard curve
of the amplicon being measured was run in duplicate ranging from 10 to 105
copies, plus a no-template control. The pNL-43 plasmid was used for the stan-
dard curve of viral DNAs. Quantitation of the human ?-globin gene was per-
formed with LightCycler-Control Kit DNA and was used to determine the
number of cells. Reactions were performed with a Light Cycler and analyzed with
the manufacturer’s software (version 3.5). Data are expressed as the numbers of
viral DNA copies per cell.
Low levels of X4 HIV replication in iDCs. To investigate the
mechanisms of HIV transmission from DCs to lymphocytes,
we first compared the replicative capacities of the R5-tropic
NLAD8 and X4-tropic NL4.3 isolates in DCs. It has been
reported that R5, and not X4, HIV-1 strains replicate in iDCs
(21, 38, 45). However, both R5 and X4 HIV-1 readily bind and
VOL. 79, 2005DC-SIGN AND HIV TRANSMISSION 5387
enter DCs (3, 13, 22, 35). We thus examined whether this
efficient capture may lead to low levels of X4 viral replication
that could have been previously underestimated. To this aim,
iDCs were derived from primary monocytes. Cells expressed
classical surface markers of bona fide iDCs (see Materials and
Methods), as well as low surface levels of CD4 and corecep-
tors, as reported previously (45). To increase the sensitivity of
detection of virus replication, iDCs were cultivated at a high
concentration (106cells/ml) and exposed to a relatively high
viral inoculum (50 ng of p24/106cells). Virus replication was
then assessed by measuring p24 production in cell superna-
tants. As expected, the R5 HIV strain replicated quite effi-
ciently, and p24 production reached 80 ng/ml at day 8 postin-
fection (p.i.) (Fig. 1A). Interestingly, under these experimental
conditions, a low but significant level of viral production was
detected with the X4 strain NL4.3, with a peak of 6 ng of
p24/ml at day 8 p.i. Using cells from six different donors, we
consistently detected viral production reaching 1 to 10 ng of
p24/ml (data not shown). This p24 production was not due to
a regurgitation of the viral inoculum, since it was inhibited by
two reverse transcriptase inhibitors, AZT (Fig. 1A) or NVP
(data not shown).
We then quantified the efficiency of RT for R5 and X4
viruses in iDCs by performing a real-time PCR analysis. iDCs
were exposed to viral inocula for 2 h (50 ng of p24/106cells),
supernatants were removed, and cells were harvested as a
function of time after initiating infection. DNA samples were
then analyzed for the presence of early (RU5) and late (pol)
FIG. 1. HIV replication and proviral DNA synthesis in monocyte-derived iDCs. (A) HIV replication. iDCs (106cells) were exposed to the
indicated viruses (50 ng of p24), with or without AZT (10 ?M). After overnight incubation, cells were washed to remove unbound virus. Viral
replication was monitored by measuring p24 production in culture supernatants. One of six independent experiments is shown. (B and C) HIV
proviral DNA synthesis. iDCs were exposed to the indicated viruses (50 ng of p24/106cells) for 3 h and extensively washed. Cell aliquots were then
immediately collected or incubated at 37°C for various periods of time. Quantification of early (RU5 DNA) (B) and late (Pol DNA) (C) viral
products was performed by real-time PCR. Data are means ? standard deviation of triplicates and are representative of at least three independent
experiments, performed with DCs from different donors.
5388 NOBILE ET AL.J. VIROL.
viral RT products (Fig. 1B). With NLAD8, we observed an
increase of total RT products over time, reaching 20 RU5
copies per cell at 24 h after infection. The late viral DNA
products were less abundant, became detectable after 10 h of
infection, and reached 3 pol copies per cell at 24 h. Viral DNA
synthesis was also observed with NL4.3, albeit at a much lower
level. After 24 h of infection, RU5 and pol copies were in the
range of 0.4 and 0.1 copies per cell, respectively. AZT was
included as a control to ensure that the detected PCR products
were the result of proviral DNA neosynthesis (Fig. 1B). Alto-
gether, these results indicate that the low levels of NL4.3 rep-
lication in iDCS, when compared to NLAD8, are associated
with a 20- to 40-fold decrease in the efficiency of the RT
We then examined whether iDCs were the major source of
virus in infected cultures. Cells were double stained with Gag
FIG. 2. Intracellular p24 expression by HIV-infected iDCs. (A) DCs from one donor were exposed to the indicated viruses (50 ng of p24/106
cells) for 2 h at 37°C. Five days later, fluorescence-activated cell sorter analysis was performed to monitor HIV infection (p24-FITC staining, x axis)
and the surface proteins DC-SIGN and MR (phycoerythrin staining, y axis). (B) iDCs from another donor were exposed to the X4 strain NL4.3
(50 ng of p24/106cells). Five days later, cells were stained for p24, DC-SIGN, MR, or CD3. Similar results were obtained with cells from four
VOL. 79, 2005DC-SIGN AND HIV TRANSMISSION5389
p24 and cell lineage markers at day 5 p.i. and analyzed by flow
cytometry (Fig. 2A). Most of the p24?cells were positive for
the DC markers DC-SIGN and MR. With NLAD8, according
to donors, between 10 to 60% of DC-SIGN?cells expressed
Gag antigens. With NL4.3, the fraction of p24?DC-SIGN?
cells varied between 0.5 to 3.5% with NL4.3 (mean of 2.2 ?
1.2%, with cells from four independent donors). DC prepara-
tions routinely contained low levels of lymphocytes (1.8 ?
1.5% of cells are CD3?, mean of four independent DC prep-
arations), which could be gated according to their size and
granularity (Fig. 2B). A subset of these low levels of CD3?
lymphocytes was productively infected by HIV (according to
donor samples, 2.7 ? 2% of CD3?cells expressed Gag p24;
mean of four experiments). Therefore, in our cell cultures,
iDCs represented the major source of Gag-expressing cells
when compared to lymphocytes (a mean of 2.2% of total cells
were Gag?DC-SIGN?, and 0.05% were Gag?CD3?). More-
over, there was no evident correlation between the percentage
of lymphocytes in a given culture and the levels of p24 pro-
duction in supernatants (data not shown). This confirmed that
most of the released virions were produced by DCs (among
Gag?cells, 98% expressed DC markers, and 2% expressed
T-cell markers). Of note, Gag p24 staining was not caused by
the viral inoculum, since it was inhibited by NVP (not shown).
FIG. 3. HIV RT in iDCs requires viral fusion. (A) Capture of WT (NL4.3) and fusion-defective (NL F522Y) HIV by iDCs. Cells were exposed
to the indicated viruses (6 or 30 ng of p24/106cells) for 2 h at 37°C and washed extensively, and cell-associated p24 was quantified by ELISA.
(B) RT in iDCs. Cells were exposed to the indicated viruses as described in the legend to Fig. 1. Quantification of RU5 viral DNA was performed
by real time PCR. (C-D) RT in HeLa cells. Cells were transiently transfected with CD4 and/or DC-SIGN and were exposed to NL4.3 (C) or
NLAD8 (D). RU5 viral products were quantified by real-time PCR as described in the legend to Fig. 1. Data are means ? standard deviation of
triplicates and are representative of at least three independent experiments.
5390 NOBILE ET AL.J. VIROL.
We concluded that the X4 HIV strain NL4.3 productively
infects, at low levels, a small fraction of iDCs.
HIV RT in DCs requires viral fusion. Both R5 and X4 HIV
virions are easily internalized by iDCs in intracellular vesicles,
in large part through binding of viral envelope glycoproteins to
DC-SIGN (9, 19, 41, 44). After capture, DC-SIGN retains
virions in an infectious state and transmits them to lympho-
cytes, with an enhancement of infection efficiency (19). We
examined whether this enhancement may be linked to the
triggering of RT in intracellular vesicles before or even without
any access of incoming virions to the cytoplasm. We thus asked
whether HIV RT in iDCs or in DC-SIGN?cell lines requires
viral fusion. To this aim, we first studied the activity of the viral
fusion inhibitor T-20 (34). T-20 inhibited proviral DNA syn-
thesis of both NL4.3 and NLAD8 strains (Fig. 1B), suggesting
that RT does not occur without viral fusion. However, T-20
was not fully active, in particular at the early time points (3 h)
of infection with NL4.3. The small amounts of proviral DNA
detected at this time may correspond to partial reverse tran-
scripts present within incoming virions (52). To further rule out
the possibility that some fusion-independent proviral DNA
synthesis events occur after viral capture, we used virions
coated with a fusion-defective HIV-1 envelope (8). This mu-
tant envelope (F522Y) retains the ability to bind CD4 (8) and
DC-SIGN (results not shown). Accordingly, both WT and mu-
tant NL4.3 F522Y virions were similarly captured by iDCs
(Fig. 3A). However, we did not detect any proviral DNA syn-
thesis by real-time PCR in cells exposed to NL4.3 F522Y (Fig.
We then documented the role of DC-SIGN during RT. We
previously reported that in HeLa cells, DC-SIGN increases the
capture and subsequent internalization of incoming virions by
about 10 fold (44). We thus used HeLa cells expressing CD4,
DC-SIGN, or both molecules as targets and followed RT upon
exposure to NL4.3. In HeLa CD4?cells, the number of total
viral DNA copies showed a transient increase, peaking at 10 h
after infection, and then declining by 24 h (Fig. 3C). This
decline has been reported previously (12) and is likely due to
the high cytopathic effect of infection in these cells. In HeLa
CD4?DC-SIGN?cells, a similar kinetic of RT was observed,
with an overall 10-fold increase in the number of RU5 copies
per cell. In the absence of CD4, DC-SIGN did not promote RT
(Fig. 3C). Viral DNA corresponded to de novo synthesis, since
the signal was dramatically decreased in the presence of NVP
(Fig. 3C). Of note, HeLa cells lack CCR5, and the R5 isolate
NLAD8 was unable to reverse transcribe, even in HeLa DC-
SIGN?CD4?cells (Fig. 3D).
Altogether, these results indicate that even though HIV
virions are efficiently captured and internalized in intracellular
vesicles in iDCs and in DC-SIGN?cell lines, RT does not
occur in the absence of a fusion event, which allows access of
incoming virions to the cytoplasm of target cells.
Absence of long-term storage of incoming infectious HIV in
iDCs. It is widely accepted that iDCs as well as some DC-
SIGN?cell lines retain competence to infect target T cells up
to 6 days after viral exposure (19, 36, 45, 54). Our findings that
the life span of incoming virions in iDCs is short (2 to 4 h) (43),
combined with our observation that X4 strains may replicate
covertly in iDCs (Fig. 1 to 3), raise the possibility that progeny
virus, rather than incoming virions, is transmitted to lympho-
cytes after prolonged periods of time. To address this question,
we exposed iDCs to various doses of single-cycle HIV-lucif-
erase pseudotyped with X4 envelope glycoproteins (HIV-Luc)
(1 to 20 ng of p24/106cells). After 2 h at 37°C, cells were
extensively washed, and virus transfer was visualized by cocul-
tivation of iDCs with target HeLa cells, expressing CD4 or not
(Fig. 4A). At 48 h later, luciferase activity in cocultures was
measured. A positive signal, increasing with the virus inocu-
lum, was detected in the presence of HeLa-CD4 cells, whereas
no signal was detected in the absence of target cells or after
iDCs were mixed with HeLa cells. These results confirmed that
iDCS are able to transfer incoming virions to target cells, if the
latter express appropriate viral receptors. We then analyzed
the ability of iDCs to retain infectious virions in the long term.
To this aim, target cells were added 48 h after iDCs were
exposed to the single-cycle reporter virus. Luciferase activity
was then measured after an additional period of 48 h (Fig. 4B).
With this time frame of 96 h, iDCs became infected and pro-
FIG. 4. Capture and transmission of incoming HIV by iDCs. iDCs
(106cells) were exposed to the indicated doses of single-cycle HIV-Luc
(pseudotyped with X4 envelope glycoproteins) and extensively washed.
Cells were then either cultured alone or with target HeLa or HeLa-
CD4 cells. Cocultures were performed either immediately (immediate
transfer) (A) or 48 h after viral exposure (long-term transfer) (B). Cell
lysates were obtained after an additional 48-h culture period and
analyzed for luciferase activity (in relative light units). Data represent
the means ? standard deviation of three separate wells of infected
cells and are representative of at least three independent experiments.
VOL. 79, 2005DC-SIGN AND HIV TRANSMISSION5391
duced luciferase, confirming that iDCs may be infected at low
levels by X4-tropic HIV. Interestingly, a signal of similar in-
tensity was detected in iDCs alone and in iDCs mixed with
HeLa-CD4 or with control HeLa cells. This strongly suggests
that only iDCs and not HeLa-CD4 cells have been infected by
HIV-Luc virions. Incoming infectious viral particles have not
been stored in iDCs during 48 h.
We then performed a time course experiment to determine
how long transmission in the absence of viral replication is able
to take place. We used as targets two T-cell lines, MT4 and
Jurkat cells, to study virus transfer from iDCs to lymphocytes.
iDCs were exposed to HIV-Luc, and target lymphocytes were
added at various time points (from 0 to 48 h). Luciferase
activity was then measured 48 h later (Fig. 5). At time zero,
HIV-Luc was efficiently transmitted from iDCs to either Jurkat
or MT4 target cells. However, a rapid decline in luciferase
activity was observed. The ability of iDCs to transfer the orig-
inal virus was reduced by half at approximately 4 to 8 h, and no
virus was transferred 24 h after exposure to DCs (Fig. 5). At a
later time point (48 h), luciferase activity in iDCs rose, and
similar levels were detected with or without T cells, confirming
that iDCs became infected at low levels, but no longer trans-
mitted the original virus inoculum to T cells.
Absence of long-term storage of incoming infectious HIV in
two DC-SIGN?B-cell lines. We further documented the role
of DC-SIGN in virus transmission. A cell-type dependent ac-
tivity of DC-SIGN has been reported (54, 58). Some cell lines,
such as Raji DC-SIGN or primary DCs, retain viral infectivity
for several days, whereas others, such as THP1 DC-SIGN,
K562 DC-SIGN, or 293 DC-SIGN, are unable to perform
these tasks. We examined whether this cell-type-dependent
effect may be linked to the ability of HIV to replicate covertly
in some cell lines and not in others. We used two B-cell lines
expressing the lectin, Raji DC-SIGN and C1RA2 DC-SIGN
(43). Cells were exposed to HIV-luciferase, and we studied
their ability to transfer infection immediately or over the long
term (after 48 h). Both Raji DC-SIGN and C1RA2 DC-SIGN
cells allowed immediate transfer of infection to target HeLa-
CD4 and not to HeLa cells (Fig. 6A). Over the long term, the
situation between these cell lines was quite different. A lucif-
erase signal was detected with Raji DC-SIGN cells in both the
absence and the presence of target cells. This situation is rem-
iniscent of that observed with primary iDCs. HIV-luciferase
productively infected Raji DC-SIGN at low levels, and no
transmission was detectable 48 h after viral exposure. In con-
trast, C1RA2 DC-SIGN cells were not detectably infected with
HIV-luciferase, and the defective virus was not transmitted
after the 48-h incubation period. Of note, similar results were
obtained when Jurkat or MT4 cells were used as targets, in-
stead of HeLa CD4 cells (not shown), confirming that Raji
DC-SIGN and C1RA2 DC-SIGN cells transfer HIV over the
short term only.
We repeated this experiment by using replicative HIV in-
stead of single-cycle virions. We reasoned that if Raji DC-
SIGN cells allow low levels of HIV replication, a long-term
transmission of replicative virus would then be detected with
these cells, whereas this should not be the case for C1RA2
DC-SIGN cells. To unambiguously detect virus transfer and
subsequent productive infection of target cells, we used HeLa-
CD4 LTR-LacZ cells (P4 cells) as targets and measured acti-
vation of the lacZ reporter gene (Fig. 6B). Both Raji DC-SIGN
and C1RA2 DC-SIGN cells transmitted infection to P4 cells
immediately after virus exposure, confirming results obtained
with single-cycle virus. Over the long term, virus transfer was
only detected with Raji DC-SIGN donor cells. Therefore, the
ability of DC-SIGN-expressing cells to retain and transfer viral
infection a few days after contact with contact correlates with
covert virus replication in these cells.
To confirm that virus replication was actually occurring in
Raji DC-SIGN cells, we measured viral DNA synthesis by
real-time PCR (Fig. 7). RU5 viral DNA was detected by 3 h p.i.
The levels of total HIV DNA increased over 24 h, reaching
0.08 copies per cell. This signal was abrogated in the presence
of NVP and was not detected in Raji cells lacking DC-SIGN
FIG. 5. Transmission of incoming HIV from iDCs to T cells. iDCs were exposed to single-cycle HIV-Luc (pseudotyped with X4 envelope
glycoproteins) (100 ng of p24/1.5 ? 106cells) and extensively washed. Cells were then either cultured alone or with target Jurkat (left) or MT4
(right) T cells. Cocultures were initiated at the indicated time points. Cell lysates were obtained after an additional 48-h culture period and analyzed
for luciferase activity (in relative light units). Data represent means ? standard deviation of three separate wells of infected cells and are
representative of three independent experiments.
5392 NOBILE ET AL.J. VIROL.
(Fig. 7). Thus, provirus DNA synthesis occurs in Raji DC-
SIGN cells at very low levels. At 24 h p.i., about 60- and
5-fold-less viral DNA was synthesized than in HeLa CD4?
DC-SIGN?or to primary iDCs, respectively (compare Fig. 3,
4, and 7). Of note, very low levels, if any, of CD4 and CCR5
receptors were detected by flow cytometry at the surface of
Raji cells, whereas CXCR4 was correctly expressed (results not
shown). We speculate that in the presence of DC-SIGN, low
levels of HIV receptors present at the cell surface or in intra-
cellular vesicles will allow fusion of incoming virions, delivery
of capsids to the cytosol, and RT.
Viral transfer from DC-SIGN?cell lines or from iDCs is pH
independent. It has been reported that DC-SIGN mediates
rapid HIV internalization into a low-pH nonlysosomal com-
partment, which allows retention and enhancement of infec-
tivity (19, 36). Moreover, neutralization of intravesicular pH by
concanamycin A, an inhibitor of vacuolar proton pump, abol-
ished the ability of Raji DC-SIGN cells to enhance virus in-
fection (36), suggesting that DC-SIGN-mediated trans en-
hancement of infection is a pH-dependent process. We
reexamined this hypothesis and asked whether viral transfer
from DC-SIGN-expressing cells or from iDCs is dependent or
independent of the intravesicular pH.
We first used Raji DC-SIGN cells as donor cells and P4
indicator cells as targets and studied the effect of concanamy-
cin A and of bafilomycin A1 on viral transfer. The latter com-
pound is another widely used proton pump inhibitor (1, 16, 40,
57). Both compounds are potent inhibitors of the virus entry
that requires acidification for fusion, like VSV. Both drugs
were rather toxic for Raji DC-SIGN cells when incubated for
long periods of time (data not shown). To reduce toxicity, Raji
DC-SIGN cells were pulse treated with either concanamycin A
(10 nM) or bafilomycin A1 (250 nM) for 1 h and for an
additional 2 h in the presence of single-cycle viruses. Cells were
then extensively washed to remove the inhibitors and incu-
bated either without or with target P4 cells for 48 h, and
luciferase activity in cell lysates was measured. We checked
that both compounds were effective in Raji DC-SIGN cells by
using HIV-luciferase virions pseudotyped with VSV-G enve-
lope glycoproteins. As expected, in the absence of P4 target
cells, direct infection of Raji DC-SIGN cells by a VSV-G-
pseudotyped HIV [HIV-Luc(VSV)] was abolished by bafilo-
mycin A1 or by concanamycin A (Fig. 8A). When a single-cycle
virus pseudotyped with HIV envelope glycoproteins was used,
we observed a moderate decrease in infection of Raji DC-
SIGN (Fig. 8B) that we attributed to moderate toxic side
FIG. 6. Capture and transmission of incoming HIV by DC-SIGN-expressing B cell lines. (A) Transmission of single-cycle HIV-Luc. Raji
DC-SIGN (top) and C1RA2 DC-SIGN (bottom) cells were exposed to the indicated doses of HIV-Luc. Cells were then either cultured alone or
with target HeLa or HeLa-CD4 cells. Cocultures were performed either immediately (immediate transfer) or 48 h after viral exposure (long-term
transfer). Cell lysates were obtained after an additional 48-h culture period and analyzed for luciferase activity. Data represent means ? standard
deviation of triplicates and are representative of at least three independent experiments. (B) Transmission of replicative HIV. Raji DC-SIGN (top)
and C1RA2 DC-SIGN (bottom) were exposed to the indicated doses of NL4.3, washed, and cocultivated with P4 cells (HeLa-CD4 LTR-LacZ
reporter cells) either immediately or 48 h after viral exposure. HIV transmission to P4 cells was assessed by measuring ?-galactosidase activity in
cell extracts after a 2-day coculture. Data (measured by optical density) are means ? standard deviation of triplicates and are representative of
three independent experiments.
VOL. 79, 2005 DC-SIGN AND HIV TRANSMISSION5393
effects of the inhibitors under these experimental conditions
(7). In the presence of P4 cells, the overall luciferase signal was
increased, confirming an immediate transfer of virions to these
cells. However, this transfer was not affected by concanamycin
A or by bafilomycin A1. This indicates that virus transmission
from Raji DC-SIGN cells is not a pH-dependent process.
It has been previously reported that at low multiplicities of
infection, activated T lymphocytes cocultivated with DCs are
more readily infected by HIV than lymphocytes directly ex-
posed to incoming virions (19, 36). We next examined the pH
dependency of the transmission of wild-type HIV from iDCs to
T lymphocytes (Jurkat cells). In our hands, concanamycin A
was rather toxic in iDCs even after extensive washing (data not
shown). We therefore employed only bafilomycin A1. When
iDCs were treated with bafilomycin A1 for 2 h and then ex-
tensively washed to remove the drug, no obvious toxicity was
observed during the following 24 to 48 h (not shown). The
compound was active in this setting, since infection with HIV-
Luc(VSV) pseudotypes was inhibited (Fig. 9A). We then used
low viral inputs (0.1 and 0.01 ng of p24/ml/106DCs) to study
the effects of bafilomycin A1 on HIV (NL4.3 strain) transmis-
sion from DCs to lymphocytes (Jurkat cells). At these multi-
plicities of infection, very low levels of virus replication, if any,
was observed in Jurkat cells alone (Fig. 9B). However, HIV
readily replicated after being in contact with DCs. Interest-
ingly, bafilomycin A1 did not affect virus growth in this system.
Thus, trans enhancement of HIV infection by DCs does not
require an acidic pH.
Altogether, these results indicate that virus transfer from
DC-SIGN?cell lines and viral trans enhancement from iDCs,
are pH-independent phenomena.
Various X4-tropic Env promote HIV replication in iDCs. It
was important to verify that the covert viral replication de-
tected in iDCs was not due to special features of NL4.3 Env
glycoproteins. We examined whether productive infection of
iDCs could be achieved using viruses that carry patient-derived
X4 envelopes. To this end, we cloned primary env genes, span-
ning the entire gp120 domain and most of gp41, into an env-
deleted NL4.3 derivative. The primary envelope sequences
were obtained by RT-PCR amplification from a plasma sample
issued from an HIV-infected patient. We designed several
replication competent viral clones, carrying envelope se-
quences issued from different variants that coexisted in the
plasma viral population (see Materials and Methods for fur-
ther details). We determined the tropism of these viral clones
by infection of U373-MG-CD4-CCR5 and U373-MG-CD4-
CXCR4 reporter cell lines (data not shown) (53). For further
studies, we selected two X4 (termed T28-X4-1 and T28-X4-2,
respectively) and one R5 (T28-R5-1) viral clones (Skrabal et
al., unpublished). iDCs (106cells/ml), were exposed to two
viral doses (50 and 5 ng of p24/106cells, respectively) for 2 h at
37°C, and viral replication was then assessed by measuring p24
production in cell supernatants. The R5 strain replicated in
iDCs, and p24 production reached 30 ng/ml at day 8 p.i. with
the larger amount of viral inoculum (Fig. 10). Interestingly,
both X4 strains replicated in iDCs, with variable efficiencies.
With T28-X4-1, viral production was detected at both high and
low inoculum levels, and reached 16 ng of p24/ml at day 10 p.i.
Replication of T28-X4-2 was much lower, peaking at 1.4 ng at
day 8 pi (Fig. 9). As for NL4.3, we detected Gag p24-expressing
DCs by fluorescence-activated cell sorter analysis (data not
shown). We conclude that a variety of X4 viruses, carrying
laboratory-adapted or primary envelope glycoproteins, pro-
ductively infect monocyte-derived iDCs at low levels.
We show here that after uptake by DCs, HIV virions can be
directly transferred to lymphocytes but only during a short
period of time (i.e., a few hours). There is no long-term storage
of captured virions by DCs or by DC-SIGN-expressing cell
lines. After a few days, virus progeny is transmitted to lympho-
We have examined the cellular and virological mechanisms
underlying these phenomena. We focused a large part of our
study on the behavior of X4 strains, which were widely believed
not being able to replicate in iDCs (14, 21, 38, 45, 60). For both
X4 and R5 isolates, the maturation state of DCs apparently
regulates viral replication. It has been reported that mature
DCs (mDCs) display a decreased capacity for the production
of HIV, which may be due to a postentry block (4, 21) and/or
to variations in CXCR4 and CCR5 expression (14, 60). What-
ever the maturation state of these cells, X4 and R5 isolates
readily enter DCs, perform RT, and are transmitted to lym-
phocytes (3, 9, 14, 22, 24, 50). It was thus puzzling that pro-
ductive infection was not detected with X4 strains in iDCs. We
provide here three lines of evidence that X4 strains replicate,
albeit at covert levels, in iDCs. First, small amounts of viral
production were measured in supernatants of cells exposed to
the laboratory-adapted NL4.3 strain, as well as two isolates
carrying primary envelope glycoproteins. Flow cytometry anal-
ysis confirmed that DCs were productively infected. Viral pro-
duction at the peak was about 10-fold lower with NL4.3 than in
the isogenic R5 strain NLAD8, which differs only in the enve-
lope gene. Second, by using a single-cycle X4 virus expressing
the luciferase reporter gene, we detected low levels of lucif-
erase expression in these cells. The viral cycle was apparently
slow, since the signal was significantly detectable at 96 h and
not at 48 h p.i. Third, quantitative PCR analysis demonstrated
FIG. 7. HIV proviral DNA synthesis in Raji DC-SIGN cells. Raji
and Raji DC-SIGN cells were exposed to NL4.3 (50 ng of p24/106cells)
as described in the legend to Fig. 1. Quantification of RU5 viral DNA
was performed by real-time PCR. Data are means ? standard devia-
tion of triplicates and are representative of three independent exper-
5394NOBILE ET AL. J. VIROL.
that NL4.3 performed RT in iDCs. Both early and late viral
DNA products were synthesized at 20 to 40-fold-lower levels
than with NLAD8. Therefore, X4 HIV strains replicate co-
vertly in monocyte-derived iDCs, and this is the consequence
of an inefficient early event of the viral cycle occurring at the
entry or postentry step.
In iDCs, as well as in DC-SIGN-expressing cell lines, a large
portion of incoming virions is internalized in intracellular ves-
icles (9, 41, 42, 44). We show here that in DCs, proviral DNA
synthesis is blocked by T-20, a viral fusion inhibitor, and does
not occur with a mutant HIV carrying nonfusogenic viral en-
velope glycoproteins. Furthermore, by using HeLa cells ex-
pressing either DC-SIGN, CD4, or both molecules, we dem-
onstrate that DC-SIGN by itself does not allow RT but
significantly enhances viral DNA synthesis in cells expressing
appropriate viral receptors (CD4 and CXCR4). Altogether,
these results indicate that efficient proviral DNA synthesis
requires access of incoming virions to the cytosol. After cap-
ture by DC-SIGN and internalization in a vesicular compart-
ment, the so-called natural endogenous RT process (61) does
not occur and is therefore not involved in the trans enhance-
ment of viral infectivity from DCs to lymphocytes.
In what form is viral infectivity is transmitted from DCs and
what is the role of DC-SIGN in this process? By using single-
cycle HIV-Luc virions, we show that incoming particles are
transferred from iDCs, as well as from Raji DC-SIGN and
C1RA2 DC-SIGN cells, to target cells immediately after viral
exposure. Parental Raji and C1RA2 cells did not transfer in-
fectivity, confirming the role of the lectin in this process. How-
ever, no increase of luciferase signal was detected if target cells
were added 48 h after viral exposure, indicating that DC-
SIGN-expressing cells do not protect virus inoculum in the
long term. Interestingly, in Raji DC-SIGN cells, a significant
luciferase activity was detected even in the absence of targets,
a situation reminiscent of primary iDCs. Low-level proviral
DNA synthesis was detected in Raji DC-SIGN cells (with a
FIG. 8. pH-independent HIV transmission by Raji DC-SIGN cells. (A) Infection by HIV(VSV) pseudotypes is inhibited by bafilomycin A1 and
concanamycin A. Raji DC-SIGN cells were pretreated or not pretreated with bafilomycin A1 (Baf A1, 250 nM) or with concanamycin A (Conc
A, 10 nM) for 1 h and then pulsed with the indicated doses of HIV-Luc pseudotyped with VSV-G [HIV-Luc(VSV)] for 2 h with or without the
drugs. Luciferase activity in cell lysates was measured 2 days later. (B) Effect of bafilomycin A1 and concanamycin A on HIV infection and
transmission by Raji DC-SIGN cells. Raji DC-SIGN cells were pretreated or not pretreated with bafilomycin A1 or concanamycin A and then
pulsed with the indicated doses of HIV-Luc as described for panel A). Cells were then grown alone (left) or cocultivated with target HeLa-CD4
cells (right). Luciferase activity was measured 2 days later in cell lysates. One out of three independent experiments is shown.
VOL. 79, 2005 DC-SIGN AND HIV TRANSMISSION5395
60-fold-lower efficiency than HeLa CD4?DC-SIGN?cells),
confirming the occurrence of surreptitious HIV replication in
these cells. Interestingly, replicative HIV and not single-cycle
virus was transmitted from Raji DC-SIGN cells 48 h after virus
exposure. Altogether, these results indicate that in the long
term, only progeny virus is transmitted from Raji DC-SIGN
cells or from iDCs to lymphocytes. Turville et al. recently
reported that DCs transfer R5 HIV to CD4?lymphocytes in
two distinct phases. By using replicative R5 HIV, they showed
that transfer of infectious virus shortly after uptake does not
require de novo synthesis, whereas the second phase of trans-
fer is inhibited by AZT and is dependent on productive infec-
tion of iDCs (57). Our experiments confirm and extend these
observations. We demonstrate here that the transfer of X4
strains follows the same rules, with an immediate phase of
transmission of incoming virions and a second phase of deliv-
ery of neosynthesized virus. Moreover, we point out that the
role of DC-SIGN is mainly at the phase of virus uptake. Most
of the incoming virions are rapidly degraded (within hours),
whereas only a small fraction reaches the contact zone when
DCs interact with T cells. DC-SIGN does not protect incoming
virions over the long term, at least in iDCs and the cell lines
studied here. The situation may be different in mDCs, which
efficiently transmit HIV in the absence of detectable produc-
tive infection (21). Upon DC maturation, DC-SIGN is down-
regulated, the endocytic capacity of the cell is decreased, and
HIV virions accumulate in vacuoles which differ in size and
intracellular localization from iDCs (18). The half-lives of in-
coming virions are also short in mDCs, but the rate of viral
decay is slightly slower and less extensive than in iDCs (57). It
is thus conceivable that mDCs retain the ability to transmit
captured virions for longer periods of time. On the other hand,
it will be worth reexamining whether productive infection oc-
curs at particularly low levels in these cells.
Our results provide a simple explanation for the cell-type-
dependent effect of DC-SIGN on long-term retention of viral
infectivity (54). We show here that Raji DC-SIGN cells share
with iDCs the capacity to replicate HIV at low levels and
FIG. 9. pH-independent HIV transmission by iDCs. (A) Infection of iDCs by HIV(VSV) pseudotypes is inhibited by bafilomycin A1. iDCs were
pretreated or not pretreated with bafilomycin A1 (Baf A1, 250 nM) for 1 h, and then pulsed with HIV-Luc(VSV) (20 ng of p24/106cells).
Luciferase activity was measured 2 days later in cell lysates. (B) Bafilomycin A1 does not affect HIV transmission from iDCs to Jurkat cells. iDCs
(5 ? 105cells) were pretreated or not pretreated for 1 h with bafilomycin A1 or mock treated, exposed to 0.1 (left) or 0.01 (right) ng of NL4./ml
for 2 h, washed, and cocultivated with Jurkat cells (5 ? 105cells). As a control, Jurkat cells were exposed to the same virus inputs and cultured
alone. Viral replication was monitored by measuring p24 production in culture supernatants. One of three independent experiments is shown.
5396 NOBILE ET AL.J. VIROL.
thereby to transfer infection to virus inoculum a few days after
the initial exposure. In other cell lines, such as HeLa DC-
SIGN, 293 DC-SIGN, or C1RA2 DC-SIGN, the absence of
productive infection precludes any retention of viral infectivity.
DC-SIGN also promotes the so-called trans enhancement of
HIV infection, through a poorly characterized mechanism.
Small amounts of virus, insufficient to allow the direct infection
of T cells, become infectious after transiting by DCs or DC-
SIGN-expressing cells (19, 36). DCs enhance infection through
the formation of an infectious synapse, which brings virus and
receptors closer together at the contact zone (41). Formation
of this synapse is important for virions in transit, but also for
transfer of newly synthesized virus particles (29) (31). On the
other hand, it has been proposed that DC-SIGN-mediated
internalization of incoming HIV is required for trans enhance-
ment (36). In this last report, trans enhancement was blocked
by concanamycin A, an inhibitor of vesicular acidification (36).
In contrast, we show here that neither concanamycin A nor
bafilomycin A1 inhibited transfer of replicative virus from
iDCs to lymphocytes or transmission of single-cycle virus from
Raji DC-SIGN to HeLa-CD4 cells. Of note, we used experi-
mental conditions (low doses, pulse incubation, and extensive
washing) to minimize the toxicity of these compounds. These
drugs are known to affect cell viability, and various side effects
have been reported that may bias the interpretation of exper-
iments aimed at raising acidic pH (7). We conclude that virus
transfer from iDCs or from DC-SIGN-expressing cell lines
does not require the low-pH environment encountered in ve-
sicular compartments. DC-SIGN-mediated enhancement has
been previously observed with single-cycle virus and thus in-
volves transfer of incoming virions (19, 36). However, our
results indicate that in donor cells where virus replicates at low
levels, such as iDCs or Raji DC-SIGN cells, a large part of the
trans enhancement process is due to the dissemination of
freshly produced virus.
R5 strains are preferentially transmitted among humans (39,
62). This restriction process is likely multifactorial and has
been suggested to take place at the stage of DC infection (49).
We show here that X4 strains replicate in iDCs, albeit at much
lower levels than R5 viruses, and are then efficiently transmit-
ted to lymphocytes. Low levels of productive X4 HIV replica-
tion have also been observed in Langerhans cell-like DCs (33,
63), as well as in more complex models of HIV dissemination,
such as ex vivo culture explants of cervical tissue (25, 28). This
should be taken into account when designing strategies aimed
at blocking HIV-1 uptake by DCs or other cells within genital
mucosa (5, 32, 48).
We thank Nathalie Sol-Foulon and Fre ´de ´ric Delebecque for critical
reading of the manuscript, Franc ¸oise Porrot and Patricia Metais-
Cunha for excellent technical help, and Eric Freed, Ali Amara, and the
NIH AIDS Research and Reference Reagent Program for the kind gift
This work was supported by grants from the Agence Nationale de
Recherche sur le SIDA (ANRS), SIDACTION, the European Com-
munity (grant QLK2-CT 2000-01630), and Institut Pasteur. C.N. is a
fellow of ANRS.
1. Aiken, C. 1997. Pseudotyping human immunodeficiency virus type 1 (HIV-1)
by the glycoprotein of VSV targets HIV-1 entry to an endocytic pathway and
suppresses both the requirement for Nef and the sensitivity to cyclosporin A.
J. Virol. 71:5871–5877.
2. Arrighi, J. F., M. Pion, M. Wiznerowicz, T. B. Geijtenbeek, E. Garcia, S.
Abraham, F. Leuba, V. Dutoit, O. Ducrey-Rundquist, Y. van Kooyk, D.
Trono, and V. Piguet. 2004. Lentivirus-mediated RNA interference of DC-
SIGN expression inhibits human immunodeficiency virus transmission from
dendritic cells to T cells. J. Virol. 78:10848–10855.
3. Ayehunie, S., E. A. Garcia-Zepeda, J. A. Hoxie, R. Horuk, T. S. Kupper, A. D.
FIG. 10. Replication of HIV strains carrying primary X4 and R5 envelope glycoproteins in iDCs. The primary envelope genes were obtained
by RT-PCR amplification from a plasma sample issued from an HIV-infected patient and cloned in an env-deleted NL4.3 HIV isolate. Two X4
(T28-X4-1 and T28-X4-2) and one R5 (T28-R5-1) isolates were analyzed. iDCs were exposed to the indicated viruses (50 or 5 ng of p24/106cells).
After overnight incubation, cells were washed to remove unbound virus. Virus replication was monitored by measuring p24 production in culture
supernatants. One of three independent experiments is shown.
VOL. 79, 2005 DC-SIGN AND HIV TRANSMISSION 5397
Luster, and R. M. Ruprecht. 1997. Human immunodeficiency virus-1 entry
into purified blood dendritic cells through CC and CXC chemokine core-
ceptors. Blood 90:1379–1386.
4. Bakri, Y., C. Schiffer, V. Zennou, P. Charneau, E. Kahn, A. Benjouad, J. C.
Gluckman, and B. Canque. 2001. The maturation of dendritic cells results in
postintegration inhibition of HIV-1 replication. J. Immunol. 166:3780–3788.
5. Baribaud, F., R. W. Doms, and S. Pohlmann. 2002. The role of DC-SIGN
and DC-SIGNR in HIV and Ebola virus infection: can potential therapeutics
block virus transmission and dissemination? Expert Opin. Ther. Targets
6. Baribaud, F., S. Pohlmann, and R. W. Doms. 2001. The role of DC-SIGN
and DC-SIGNR in HIV and SIV attachment, infection, and transmission.
7. Bayer, N., D. Schober, E. Prchla, R. F. Murphy, D. Blaas, and R. Fuchs.
1998. Effect of bafilomycin A1 and nocodazole on endocytic transport in
HeLa cells: implications for viral uncoating and infection. J. Virol. 72:9645–
8. Bergeron, L., N. Sullivan, and J. Sodroski. 1992. Target cell-specific deter-
minants of membrane fusion within the human immunodeficiency virus type
1 gp120 third variable region and gp41 amino terminus. J. Virol. 66:2389–
9. Blauvelt, A., H. Asada, M. W. Saville, V. Klaus Kovtun, D. J. Altman, R.
Yarchoan, and S. I. Katz. 1997. Productive infection of dendritic cells by
HIV-1 and their ability to capture virus are mediated through separate
pathways. J. Clin. Investig. 100:2043–2053.
10. Brussel, A., and P. Sonigo. 2003. Analysis of early human immunodeficiency
virus type 1 DNA synthesis by use of a new sensitive assay for quantifying
integrated provirus. J. Virol. 77:10119–10124.
11. Buseyne, F., S. Le Gall, C. Boccaccio, J. P. Abastado, J. D. Lifson, L. O.
Arthur, Y. Rivie `re, J. M. Heard, and O. Schwartz. 2001. MHC-I-restricted
presentation of HIV-1 virion antigens without viral replication. Nat. Med.
12. Butler, S. L., E. P. Johnson, and F. D. Bushman. 2002. Human immunode-
ficiency virus cDNA metabolism: notable stability of two-long terminal re-
peat circles. J. Virol. 76:3739–3747.
13. Cameron, P., M. Pope, A. Granelli-Piperno, and R. M. Steinman. 1996.
Dendritic cells and the replication of HIV-1. J. Leukoc. Biol. 59:158–171.
14. Canque, B., Y. Bakri, S. Camus, M. Yagello, A. Benjouad, and J. C. Gluck-
man. 1999. The susceptibility to X4 and R5 human immunodeficiency virus-1
strains of dendritic cells derived in vitro from CD34?hematopoietic pro-
genitor cells is primarily determined by their maturation stage. Blood 93:
15. Connor, R. I., B. K. Chen, S. Choe, and N. R. Landau. 1995. Vpr is required
for efficient replication of human immunodeficiency virus type-1 in mono-
nuclear phagocytes. Virology 206:935–944.
16. Drose, S., and K. Altendorf. 1997. Bafilomycins and concanamycins as in-
hibitors of V-ATPases and P-ATPases. J. Exp. Biol. 200:1–8.
17. Feinberg, H., D. A. Mitchell, K. Drickamer, and W. I. Weis. 2001. Structural
basis for selective recognition of oligosaccharides by DC-SIGN and DC-
SIGNR. Science 294:2163–2166.
18. Frank, I., M. Piatak, Jr., H. Stoessel, N. Romani, D. Bonnyay, J. D. Lifson,
and M. Pope. 2002. Infectious and whole inactivated simian immunodefi-
ciency viruses interact similarly with primate dendritic cells (DCs): differen-
tial intracellular fate of virions in mature and immature DCs. J. Virol.
19. Geijtenbeek, T. B., D. S. Kwon, R. Torensma, S. J. Van Vliet, G. C. Van
Duijnhoven, J. Middel, I. L. Cornelissen, H. Nottet, V. KewalRamani, D.
Littman, C. G. Figdor, and Y. Van Kooyk. 2000. DC-SIGN, a dendritic
cell-specific HIV-1-binding protein that enhances trans-infection of T cells.
20. Goxe, B., N. Latour, J. Bartholeyns, J. L. Romet-Lemonne, and M. Chokri.
1998. Monocyte-derived dendritic cells: development of a cellular processor
for clinical applications. Res. Immunol. 149:643–646.
21. Granelli-Piperno, A., E. Delgado, V. Finkel, W. Paxton, and R. M. Steinman.
1998. Immature dendritic cells selectively replicate macrophagetropic (M-
tropic) human immunodeficiency virus type 1, while mature cells efficiency
transmit both M- and T-tropic virus to T cells. J. Virol. 72:2733–2737.
22. Granelli-Piperno, A., V. Finkel, E. Delgado, and R. M. Steinman. 1999. Virus
replication begins in dendritic cells during the transmission of HIV-1 from
mature dendritic cells to T cells. Curr. Biol. 9:21–29.
23. Granelli-Piperno, A., A. Golebiowska, C. Trumpfheller, F. P. Siegal, and
R. M. Steinman. 2004. HIV-1-infected monocyte-derived dendritic cells do
not undergo maturation but can elicit IL-10 production and T cell regulation.
Proc. Natl. Acad. Sci. USA 101:7669–7674.
24. Granelli-Piperno, A., B. Moser, M. Pope, D. Chen, Y. Wei, F. Isdell, U.
O’Doherty, W. Paxton, R. Koup, S. Mojsov, N. Bhardwaj, I. Clark-Lewis, M.
Baggiolini, and R. M. Steinman. 1996. Efficient interaction of HIV-1 with
purified dendritic cells via multiple chemokine coreceptors. J. Exp. Med.
25. Greenhead, P., P. Hayes, P. S. Watts, K. G. Laing, G. E. Griffin, and R. J.
Shattock. 2000. Parameters of human immunodeficiency virus infection of
human cervical tissue and inhibition by vaginal virucides. J. Virol. 74:5577–
26. Gummuluru, S., V. N. KewalRamani, and M. Emerman. 2002. Dendritic
cell-mediated viral transfer to T cells is required for human immunodefi-
ciency virus type 1 persistence in the face of rapid cell turnover. J. Virol.
27. Gummuluru, S., M. Rogel, L. Stamatatos, and M. Emerman. 2003. Binding
of human immunodeficiency virus type 1 to immature dendritic cells can
occur independently of DC-SIGN and mannose binding C-type lectin recep-
tors via a cholesterol-dependent pathway. J. Virol. 77:12865–12874.
28. Hu, Q., I. Frank, V. Williams, J. J. Santos, P. Watts, G. E. Griffin, J. P.
Moore, M. Pope, and R. J. Shattock. 2004. Blockade of attachment and
fusion receptors inhibits HIV-1 infection of human cervical tissue. J. Exp.
29. Igakura, T., J. C. Stinchcombe, P. K. Goon, G. P. Taylor, J. N. Weber, G. M.
Griffiths, Y. Tanaka, M. Osame, and C. R. Bangham. 2003. Spread of
HTLV-I between lymphocytes by virus-induced polarization of the cytoskel-
eton. Science 299:1713–1716.
30. Jameson, B., F. Baribaud, S. Pohlmann, D. Ghavimi, F. Mortari, R. W.
Doms, and A. Iwasaki. 2002. Expression of DC-SIGN by dendritic cells of
intestinal and genital mucosae in humans and rhesus macaques. J. Virol.
31. Jolly, C., K. Kashefi, M. Hollinshead, and Q. J. Sattentau. 2004. HIV-1 cell
to cell transfer across an Env-induced, actin-dependent synapse. J. Exp.
32. Kawamura, T., S. S. Cohen, D. L. Borris, E. A. Aquilino, S. Glushakova, L. B.
Margolis, J. M. Orenstein, R. E. Offord, A. R. Neurath, and A. Blauvelt.
2000. Candidate microbicides block HIV-1 infection of human immature
Langerhans cells within epithelial tissue explants. J. Exp. Med. 192:1491–
33. Kawamura, T., M. Qualbani, E. K. Thomas, J. M. Orenstein, and A. Blau-
velt. 2001. Low levels of productive HIV infection in Langerhans cell-like
dendritic cells differentiated in the presence of TGF-?1 and increased viral
replication with CD40 ligand-induced maturation. Eur. J. Immunol. 31:360–
34. Kilby, J. M., S. Hopkins, T. M. Venetta, B. DiMassimo, G. A. Cloud, J. Y.
Lee, L. Alldredge, E. Hunter, D. Lambert, D. Bolognesi, T. Matthews, M. R.
Johnson, M. A. Nowak, G. M. Shaw, and M. S. Saag. 1998. Potent suppres-
sion of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-
mediated virus entry. Nat. Med. 4:1302–1307.
35. Klagge, I. M., and S. Schneider-Schaulies. 1999. Virus interactions with
dendritic cells. J. Gen. Virol. 80:823–833.
36. Kwon, D. S., G. Gregorio, N. Bitton, W. A. Hendrickson, and D. R. Littman.
2002. DC-SIGN-mediated internalization of HIV is required for trans-en-
hancement of T cell infection. Immunity 16:135–144.
37. Le Gall, S., L. Erdtmann, S. Benichou, C. Berlioz-Torrent, L. X. Liu, R.
Benarous, J. M. Heard, and O. Schwartz. 1998. Nef interacts with ? subunits
of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC-I
molecules. Immunity 8:483–495.
38. Lin, C. L., A. K. Sewell, G. F. Gao, K. T. Whelan, R. E. Phillips, and J. M.
Austyn. 2000. Macrophage-tropic HIV induces and exploits dendritic cell
chemotaxis. J. Exp. Med. 192:587–594.
39. Liu, R., W. A. Paxton, S. Choe, D. Ceradini, S. R. Martin, R. Horuk, M. E.
MacDonald, H. Stuhlmann, R. A. Koup, and N. R. Landau. 1996. Homozy-
gous defect in HIV-1 coreceptor accounts for resistance of some multiply-
exposed individuals to HIV-1 infection. Cell 86:367–377.
40. Mare ´chal, V., F. Clavel, J. M. Heard, and O. Schwartz. 1998. Cytosolic Gag
p24 as an index of productive entry of human immunodeficiency virus type 1.
J. Virol. 72:2208–2212.
41. McDonald, D., L. Wu, S. M. Bohks, V. N. KewalRamani, D. Unutmaz, and
T. J. Hope. 2003. Recruitment of HIV and its receptors to dendritic cell-T
cell junctions. Science 300:1295–1297.
42. Messmer, D., R. Ignatius, C. Santisteban, R. M. Steinman, and M. Pope.
2000. The decreased replicative capacity of simian immunodeficiency virus
SIVmac239?nef is manifest in cultures of immature dendritic cells and T
cells. J. Virol. 74:2406–2413.
43. Moris, A., C. Nobile, F. Buseyne, F. Porrot, J. P. Abastado, and O. Schwartz.
2004. DC-SIGN promotes exogenous MHC-I-restricted HIV-1 antigen pre-
sentation. Blood 103:2648–2654.
44. Nobile, C., A. Moris, F. Porrot, N. Sol-Foulon, and O. Schwartz. 2003.
Inhibition of human immunodeficiency virus type 1 Env-mediated fusion by
DC-SIGN. J. Virol. 77:5313–5323.
45. Petit, C., F. Buseyne, C. Boccaccio, J. P. Abastado, J. M. Heard, and O.
Schwartz. 2001. Nef is required for efficient HIV-1 replication in cocultures
of dendritic cells and lymphocytes. Virology 286:225–236.
46. Pope, M., M. G. Betjes, N. Romani, H. Hirmand, P. U. Cameron, L. Hoff-
man, S. Gezelter, G. Schuler, and R. M. Steinman. 1994. Conjugates of
dendritic cells and memory T lymphocytes from skin facilitate productive
infection with HIV-1. Cell 78:389–398.
47. Pope, M., S. Gezelter, N. Gallo, L. Hoffman, and R. M. Steinman. 1995. Low
levels of HIV-1 infection in cutaneous dendritic cells promote extensive viral
5398 NOBILE ET AL.J. VIROL.
replication upon binding to memory CD4? T cells. J. Exp. Med. 182:2045– Download full-text
48. Pope, M., and A. T. Haase. 2003. Transmission, acute HIV-1 infection and
the quest for strategies to prevent infection. Nat Med. 9:847–852.
49. Reece, J. C., A. J. Handley, E. J. Anstee, W. A. Morrison, S. M. Crowe, and
P. U. Cameron. 1998. HIV-1 selection by epidermal dendritic cells during
transmission across human skin. J. Exp Med. 187:1623–1631.
50. Sanders, R. W., E. C. de Jong, C. E. Baldwin, J. H. Schuitemaker, M. L.
Kapsenberg, and B. Berkhout. 2002. Differential transmission of human
immunodeficiency virus type 1 by distinct subsets of effector dendritic cells.
J. Virol. 76:7812–7821.
51. Schwartz, O., J. L. Virelizier, L. Montagnier, and U. Hazan. 1990. A micro-
transfection method using luciferase gene for the study of human immuno-
deficiency virus long terminal repeat activity. Gene 88:197–205.
52. Trono, D. 1992. Partial reverse transcripts in virions from human immuno-
deficiency and murine leukemia viruses. J. Virol. 66:4893–4900.
53. Trouplin, V., F. Salvatori, F. Cappello, V. Obry, A. Brelot, N. Heveker, M.
Alizon, G. Scarlatti, F. Clavel, and F. Mammano. 2001. Determination of
coreceptor usage of human immunodeficiency virus type 1 from patient
plasma samples by using a recombinant phenotypic assay. J. Virol. 75:251–
54. Trumpfheller, C., C. G. Park, J. Finke, R. M. Steinman, and A. Granelli-
Piperno. 2003. Cell type-dependent retention and transmission of HIV-1 by
DC-SIGN. Int. Immunol. 15:289–298.
55. Turville, S. G., J. Arthos, K. M. Donald, G. Lynch, H. Naif, G. Clark, D.
Hart, and A. L. Cunningham. 2001. HIV gp120 receptors on human den-
dritic cells. Blood 98:2482–2488.
56. Turville, S. G., P. U. Cameron, A. Handley, G. Lin, S. Po ¨hlmann, R. W.
Doms, and A. L. Cunningham. 2002. Diversity of receptors binding HIV on
dendritic cell subsets. Nat. Immunol. 3:975–983.
57. Turville, S. G., J. J. Santos, I. Frank, P. U. Cameron, J. Wilkinson, M.
Miranda-Saksena, J. Dable, H. Stossel, N. Romani, M. Piatak, Jr., J. D.
Lifson, M. Pope, and A. L. Cunningham. 2004. Immunodeficiency virus
uptake, turnover, and 2-phase transfer in human dendritic cells. Blood 103:
58. Wu, L., T. D. Martin, M. Carrington, and V. N. KewalRamani. 2004. Raji B
cells, misidentified as THP-1 cells, stimulate DC-SIGN-mediated HIV trans-
mission. Virology 318:17–23.
59. Wu, L., T. D. Martin, Y. C. Han, S. K. Breun, and V. N. KewalRamani. 2004.
Trans-dominant cellular inhibition of DC-SIGN-mediated HIV-1 transmis-
sion. Retrovirology 1:14.
60. Zaitseva, M., A. Blauvelt, S. Lee, C. K. Lapham, V. Klaus-Kovtun, H.
Mostowski, J. Manischewitz, and H. Golding. 1997. Expression and function
of CCR5 and CXCR4 on human Langerhans cells and macrophages: impli-
cations for HIV primary infection. Nat. Med. 3:1369–1375.
61. Zhang, H., G. Dornadula, and R. J. Pomerantz. 1996. Endogenous reverse
transcription of human immunodeficiency virus type 1 in physiological mi-
croenviroments: an important stage for viral infection of nondividing cells.
J. Virol. 70:2809–2824.
62. Zhu, T., H. Mo, N. Wang, D. S. Nam, Y. Cao, R. A. Koup, and D. D. Ho. 1993.
Genotypic and phenotypic characterization of HIV-1 patients with primary
infection. Science 261:1179–1181.
63. Zoeteweij, J. P., H. Golding, H. Mostowski, and A. Blauvelt. 1998. Cytokines
regulate expression and function of the HIV coreceptor CXCR4 on human
mature dendritic cells. J. Immunol. 161:3219–3223.
VOL. 79, 2005 DC-SIGN AND HIV TRANSMISSION5399