Rab5 and Rab7, but Not ARF6, Govern the
Early Events of HIV-1 Infection in Polarized
Human Placental Cells
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J Immunol 2005; 175:6517-6530; ;
doi: 10.4049/jimmunol.175.10.6517
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References
Gaël Vidricaire and Michel J. Tremblay
The Journal of Immunology
Rab5 and Rab7, but Not ARF6, Govern the Early Events of
HIV-1 Infection in Polarized Human Placental Cells1
Gaël Vidricaire and Michel J. Tremblay2
V
ertical transmission of the HIV type 1 (HIV-1)3 is a serious global health problem. Currently, 2.1 millions children ⬍ 15 years of age are infected by HIV-1 worldwide
and 90% of these infections are associated with mother-to-child
transmission (MTCT) of this retrovirus (1). The risk of vertical
transmission lies within the range of 15–25% in industrialized
countries and reaches 35% in developing countries (1). MTCT
may occur in utero, at birth during delivery, or through breastfeeding (reviewed in Ref. 2). Vertical transmission occurring through
breastfeeding or at birth most likely involves ingestion of contaminated fluids (e.g., milk, vaginal secretion, and blood) by the infant.
In contrast, in utero HIV-1 transmission requires a direct implication of the placenta (reviewed in Ref. 2).
Mathematical modeling has estimated that while 65% of the
infections occur at birth, one-third of the infants are considered to
become infected in utero ⬍2 mo before delivery (3). This being
Research Center in Infectious Diseases, Centre Hospitalier de l’Université Laval Research Center, and Faculty of Medicine, Laval University, Quebec City, Quebec,
Canada
Received for publication February 8, 2005. Accepted for publication September
1, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Canadian Institutes of Health Research (CIHR) HIV/
AIDS Research Program Grant (HOP-67259) (to M.J.T.). The work was performed
by G.V. in partial fulfillment of her Ph.D. degree in the Program of MicrobiologyImmunology, Faculty of Medicine, Laval University. G.V. is the recipient of a CIHR
Doctoral Research Award from the HIV/AIDS Research Program, and M.J.T. holds
the Canada Research Chair in Human Immuno-Retrovirology (tier 1 level).
2
Address correspondence and reprint requests to Dr. Michel J. Tremblay, Laboratory
of Human Immuno-Retrovirology, Research Center in Infectious Diseases, RC709,
CHUL Research Center, 2705 Laurier Boulevard, Quebec City, Quebec G1V 4G2,
Canada. E-mail address: [email protected]
3
Abbreviations used in this paper: HIV-1, HIV type 1; MTCT, mother-to-child transmission; Env, envelope; VSV-G, vesicular stomatitis virus Env glycoprotein G;
EGFP, enhanced GFP; AZT, zidovudine; RLU, relative light unit; LTR, long terminal
repeat.
Copyright © 2005 by The American Association of Immunologists, Inc.
said, in utero HIV-1 transmission has been documented by several
groups (4 –7). In addition, vertical transmission can occur early
during pregnancy because HIV-1 has been detected in aborted fetuses as early as after 8 wk of gestation (8) and in first and second
trimester fetuses (9 –12). In utero transmission of HIV-1 is poorly
understood, and the importance of studying such a process is related to the following facts. If we consider again the mathematical
modeling cited above, it can be estimated that among the 2 million
children currently living with HIV-1/AIDS, hundreds of thousands
of these infections occurred in utero. Hence, although in utero
transmission is unlikely the primary cause of infection in infants,
given the scale of the pandemic the resulting numbers are substantial. Additionally, the risks of acquiring HIV-1 is 1.3-fold higher in
adult African women than in men of the same age, and among
young women aged between 15 and 24 years, this disparity is more
important because a 3-fold difference is reported. As a result,
women are becoming infected at a higher frequency than men (13,
14). Antiretroviral therapies are known to significantly reduce the
incidence of vertical transmission (15). However, according to recent statistics gathered in developing countries, only 10% of pregnant women received prebirth counseling for preventing MTCT
and only 7% of people had access to antiretroviral treatments in
2003 (13, 14). Consequently, initiatives to prevent vertical transmission of HIV-1 are now aimed at developing strategies that are
readily available to women. In this context, it is well recognized
that a better understanding of how and when vertical transmission
occurs is crucial (13, 14).
The placenta functions as a barrier composed of a double layer
of polarized epithelial-type cells, i.e., the cytotrophoblasts and syncytiotrophoblasts. Trophoblasts separate the maternal and fetal
blood circulations and control fluxes between them. Thus, HIV-1
must first cross this natural barrier before reaching the fetal circulation. Current models propose that following infection of trophoblasts and/or transcytosis across these cells, HIV-1 is released from
the basolateral pole of the trophoblasts (facing the fetal circulation)
leading to productive infection of the underlying fetal cells (16 –
0022-1767/05/$02.00
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Trophoblasts, the structural cells of the placenta, are thought to play a determinant role in in utero HIV type 1 (HIV-1) transmission. We have accumulated evidence suggesting that HIV-1 infection of these cells is associated with uptake by an unusual
clathrin/caveolae-independent endocytic pathway and that endocytosis is followed by trafficking through multiple organelles.
Furthermore, part of this trafficking involves the transit of HIV-1 from transferrin-negative to EEA1 and transferrin-positive
endosomes, suggesting a merger from nonclassical to classical endocytic pathways in these cells. In the present article, the relationship between the presence of HIV-1 within specific endosomes and infection was studied. We demonstrate that viral infection
is virtually lost when endosome inhibitors are added shortly after exposure to HIV-1. Thus, contrary to what is seen in CD4ⴙ T
lymphocytes, the initial presence of HIV-1 within the endosomes is mandatory for infection to take place. Importantly, this process
is independent of the viral envelope proteins gp120 and gp41. The Rab family of small GTPases coordinates the vesicular transport
between the different endocytic organelles. Experiments performed with various expression vectors indicated that HIV-1 infection
in polarized trophoblasts relies on Rab5 and Rab7 without the contribution of Arf6 or Rab11. Furthermore, we conclude that
Rab5 drives movements from raft-rich region to early endosomes, and this transit is required for subsequently reaching late
endosomes via Rab7. This complex trafficking is mandatory for HIV-1 infection to proceed in human polarized trophoblasts. The
Journal of Immunology, 2005, 175: 6517– 6530.
6518
ENDOCYTOSIS OF HIV-1 IN HUMAN POLARIZED TROPHOBLASTS
Dominant-negative or active mutant versions of Rab proteins have
proven to be powerful tools for analyzing defined trafficking pathways
within cells (reviewed in Ref. 37). The dominant-negative Rab5 mutant (Rab5 S34N), defective in guanine-nucleotide binding, inhibits
early endosome fusion and causes the accumulation of small vesicles
at the cell periphery. By contrast, the constitutively active Rab5 mutant (Rab5 Q79L) enhances endocytosis and early endosome fusion,
causing the formation of enlarged early endosomes. Similar observations are obtained upon overexpression of wild-type Rab5 (38). The
dominant-negative Rab7 mutant (Rab7 T22N) blocks the transport of
receptors to late endosomes and results in its retention in Rab5-positive early endosomes. Cells expressing a wild-type or a constitutively
active Rab7 mutant (Rab7 Q67L) exhibit enhanced association with
lysosomes (39, 40). The Rab11 mutant defective in GTP binding
(Rab11 S25N) impairs recycling by inhibiting transport from sorting
endosomes (early endosomes) to the recycling endosomes (41). Overexpression of wild-type Rab11 or dominant-active Rab11 (Rab11
Q70L) promotes recycling in some instances (42).
An important issue arises for macromolecules/pathogens internalized independently of clathrin-coated pits. Indeed, these macromolecules may or may not intersect with the classical endocytic
pathway, and if indeed they do, the mechanism underlying such an
event is poorly understood. The Ras-related protein Arf6 can return glycosylphosphatidylinositol-anchored proteins to the plasma
membrane. Alternatively, inactivation of Arf6 leads to convergence with the classical early endosomes and traffic to late endosomes/lysosomes (43).
As described earlier, the contact between HIV-1 and trophoblasts results in massive and rapid uptake of virions within these
cells by endocytosis, a process that is required for infection to
proceed in trophoblasts (27, 33). We have recently provided evidence that the internalization pathway leading to HIV-1 infection
of trophoblasts is independent of clathrin-coated pits and caveolae
but is sensitive to raft-perturbing drugs (G. Vidricaire and M. J.
Tremblay, submitted for publication). Moreover, the early events
associated with HIV-1 infection in polarized human trophoblasts
involve an active participation of the endocytic machinery (33).
For instance, confocal studies demonstrated that although HIV-1
particles do not initially colocalize with transferrin in polarized
JAR cells, a marker of the classical endocytic pathway, they do at
later time points (G. Vidricaire and M. J. Tremblay, submitted for
publication). Moreover, we found that HIV-1 is in part associated
with EEA1-expressing early endosomes (33). Given that HIV-1
internalization does not rely on clathrin-mediated endocytosis, the
taken intracellular endocytic route most likely involves a merger
between nonclassical endocytic and classical pathways (G. Vidricaire and M. J. Tremblay, submitted for publication). In contrast,
with time, some virions also reach Rab11⫹ recycling endosomes.
However, the majority of the virions accumulate within CD63⫹
late endosomes (33). Given that HIV-1 is located within different
organelles upon internalization into trophoblasts and its presence
within endosomes is necessary for infection to ensue (33), defining
which organelle(s) is required for infection to proceed in polarized
trophoblasts might provide critical information on in utero HIV-1
transfer. In this study, the involvement of specific Rab proteins
(i.e., Rab5, Rab7, and Rab11) and Arf6 was analyzed in the context of viral infection in polarized trophoblasts. We found an intriguing relationship between trafficking of HIV-1 within the endocytic compartments and viral infection. Collectively, our data
provide unprecedented information on the identity of subcellular
organelles that might be involved in vertical transmission of
HIV-1.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
23). The mechanisms associated with these events are ill-defined,
and it is also unknown in which proportions each pathway contributes to vertical transmission.
HIV-1 is an enveloped virus that gains entry into susceptible
cells upon fusion of its outer membrane with the plasma membrane
of a target cell. This process involves coordinated interactions between the viral envelope (Env) glycoproteins gp120 and gp41 and
the primary cellular receptor CD4 and a coreceptor such as
CXCR4 or CCR5. HIV-1 infection is pH independent and does not
require endocytosis of CD4 (24, 25). Several reports have documented that trophoblasts sustain HIV-1 infection both in vitro and
in vivo (16, 17, 19 –23, 26, 27). However, as with other epithelial
cells, macrophages, dendritic cells, and astrocytes, their permissiveness to HIV-1 infection is estimated to be lower than that of
CD4⫹ T lymphocytes, the primary target of HIV-1 infection (our
unpublished observations and Ref. 27). One of the reasons postulated for such a restricted susceptibility to virus infection is linked
with the low to absent expression of CD4, CXCR4, and CCR5 in
trophoblasts (28 –32). At the same time, the process of viral entry
is enigmatic given the absence of the appropriate receptor and
coreceptors in these cells in spite of a moderate productive infection. Contrary to previous assumptions, we made the surprising
discovery that HIV-1 is massively endocytosed within trophoblastic cells (27, 33). However, as a consequence, virions are predominantly trapped within the endosomal compartments of the cells.
This event is believed to translate into weak HIV-1 production due
to an inefficient endosomal escape by HIV-1 combined to a rapid
degradation of the majority of the viral particles in the endosomal/
lysosomal compartments of the infected cells. Because of this entry pathway, although trophoblasts display no blocking mechanism
for viral transcription or viral production, proinflammatory cytokines are required to promote virus gene expression in these cells.
It is postulated that such cytokines, transiently expressed during
pregnancy, sporadically create favorable conditions leading to in
vivo vertical transmission of this retrovirus (21, 27, 31). It is important to state that virus entry leading to infection of trophoblasts
is unexpectedly independent of gp120 and gp41 (G. Vidricaire and
M. J. Tremblay, submitted for publication). Finally, inhibitors of
the function of the endosomes block HIV-1 infection of trophoblasts (33). This last observation is particularly meaningful because it implies that internalization of HIV-1 by endocytosis,
which results in the presence of viral material within the endosomes, is an obligatory step for infection to proceed in human
trophoblasts. This is radically different from what is seen in CD4⫹
T lymphocytes (24, 25, 34). Given that internalization of HIV-1
within trophoblasts is required for infection to take place in these
cells, defining how the presence of HIV-1 within the endocytic
compartments relates to infection in trophoblastic cells is crucial.
This article addresses this key question.
Upon internalization, macromolecules (or pathogens) travel
through a number of endocytic subcompartments where sorting
decisions are made for recycling to the plasma membrane or targeting to the lumen of late endosomes/lysosomes. Hence, the fate
of macromolecules/pathogens is dictated by their intracellular endocytic route. Importantly, the precise location in endocytic compartments where viral escape is occurring is often critical in yielding productive infection or not (35). The Rab family of small
GTPases coordinates the vesicular transport between the different
endocytic organelles. Distinct Rab proteins have been identified
and each is specifically associated with a particular organelle or
pathway. For instance, Rab5 is needed for early endosomes, Rab7 for
transport from early to late endosomes, and from late endosomes to
lysosomes, whereas Rab11 is enriched in recycling endosomes (36).
The Journal of Immunology
Materials and Methods
Cells
The human trophoblastic JAR and 293T cell lines were obtained from the
American Type Culture Collection. Cells were maintained as previously
described (33). For the experiments performed in this work, JAR cells were
grown into a polarized monolayer. This goal was achieved by seeding JAR
cells in 24-well cell culture inserts containing a 0.4-␮m pore size polyethylene terephthalate track-etched membrane (BD Biosciences Labware),
and the cells were grown to complete confluence and polarize for 4 days as
described previously (33).
6519
mating kinetics of inhibition of HIV-1 infection, bafilomycin A1 or NH4Cl
was added 30 min before virus exposure for 6 h at 37°C. Cells were next
washed twice with PBS, and fresh medium was added. Bafilomycin A1 or
NH4Cl was added at this point (i.e., 6 h after the onset of infection) or 12
or 24 h following the onset of virus infection. The proinflammatory cytokine TNF-␣ (10 ng/ml) was also added at 6 h following the initial exposure
to HIV-1. Finally, the cells were lysed 24 h later, and luciferase activity,
which is indicative of virus gene expression, was monitored in each cell
lysate.
Cell fractionation assay
Transfection of polarized JAR cells
Real-time PCR
The cDNAs of the small GTPase proteins and their mutants are all fusion
proteins with the enhanced GFP (EGFP). The EGFP, EGFP-Rab5 (i.e.,
Rab5 WT, Rab5 S34N, and Rab5 Q79L) and EGFP-Rab11 expression
vectors (i.e., Rab11 WT, Rab11 S25N, and Rab11 Q70L) were provided by
S. S. Ferguson (Robarts Research Institute, London, Ontario, Canada). The
EGFP-Rab7 expression vectors (i.e., Rab7 Q67L and Rab7 T22N) were
supplied by B. van Deurs (The Panum Institute, University of Copenhagen,
Copenhagen, Denmark), and the EGFP-Arf6 expression vectors (i.e., Arf6
WT, Arf6 T27N and Arf6 Q67L) were gifts from J. G. Donaldson (National Institutes of Health, Bethesda, MD). Polarized JAR cells (as described above) were transfected using Lipofectamine (Invitrogen Canada).
Cells were either mock-transfected or transfected with 0.2 ␮g of carrier
DNA and 5, 6, 15, or 25 ng of the expression vector of interest. The
following day, cells were washed three times with PBS. Viral entry and
infection assays were performed 12–24 h later as described below. Transfection efficiencies when using the various Rab expression vectors reached
50% as monitored by EGFP expression (data not shown).
Polarized JAR and Jurkat cells (1 ⫻ 106 cells) were either left untouched
or exposed to NL4-3 wild-type viruses (250 ng of p24) in the absence or
in the presence of AZT (10 ␮M) for 48 h at 37°C. Infected cells were then
washed twice with PBS and trypsinized at 37°C for 5 min. The cells were
pelleted down by centrifugation (1200 rpm for 5 min at 4°C) and washed
twice with PBS. Total cellular DNA was isolated using the DNeasy Tissue
kit as described by the manufacturer (Qiagen). To quantify the amount of
integrated viral DNA, we used the real-time PCR approach described by
Suzuki et al. (48). Briefly, a first round of PCR amplification was conducted using 100 ng of DNA and TaqDNA polymerase (Promega). The
primers were as follows: Alu-specific sense primer (5⬘-TCC CAG CTA
CTC GGG AGG CTG AGG-3⬘) and an antisense HIV-1-specific primer
(M661) (5⬘-CCT GCG TCG AGA GAT CTC CTC TG-3⬘, 673– 695). Cycling conditions included an initial denaturation step (94°C for 3 min),
followed by 22 cycles of denaturation (94°C for 30s), annealing (66°C for
30s), and extension (70°C for 10 min) followed by a final extension (72°C
for 10 min). The first PCR products were subsequently diluted 5-fold and
subjected to a real-time PCR assay targeting the HIV-1 R/U5 region. The
primers were as follows: R/U5 DNA sense primer (M667) (5⬘-GGC TAA
CTA GGG AAC CCA CTG C-3⬘, 496 –517) and the antisense primer
(AA55) (5⬘-CTG CTA GAG ATT TTC CAC ACT GAC-3⬘, 612– 635).
The fluorogenic probe HIV-FAM (Biosearch Technologies) was designed
to anneal to the target between the sense primer M667 and the antisenseprimer AA55 in the R-U5 region: 5⬘-(FAM) TAG TGT GTG CCC GTC
TGT TGT GTG AC (BHQ-1)-3⬘. PCR was performed using the RotorGene 3000 four-channel multiplexing system (Corbett Research) and
TaqDNA polymerase. Cycling conditions included 40 cycles of denaturation (95°C for 20 s) and extension (60°C for 1 min). To detect retrotranscribed viral DNA, isolated DNA was subjected to the first PCR in the
absence of TaqDNA polymerase and then the samples were subjected to
the real-time PCR.
Virus entry assay
Polarized JAR cells were pretreated with vehicle, 0.1 ␮M bafilomycin A1,
10 mM ammonium chloride (NH4Cl), or 10 nM wortmannin (Sigma-Aldrich) for 30 min at 37°C. These pretreated cells or the transiently transfected JAR cells were exposed to pseudotyped and Env-deficient viruses
(250 ng of p24) for 1 h at 37°C. At this point, the cells were acid-treated
(0.5 M NaCl and 1% acetic acid) at 4°C for 1 min and washed three times
with ice-cold PBS to remove all uninternalized viral particles. Finally, the
cells were lysed in ice-cold lysis buffer (20 mM HEPES (pH 7.4), 150 mM
NaCl, and 0.5% Triton X-100), and virus content was measured using a
homemade p24 enzymatic test (46).
Infection assay
Polarized JAR or Jurkat cells (1 ⫻ 106 cells) were exposed to recombinant
luciferase-encoding viruses pseudotyped with HXB-2 (0, 100, 125, and 250
ng of p24 or 250 ng together with 10 ␮M zidovudine (AZT)) at 37°C for
24 h. In some experiments, untransfected or transiently transfected polarized JAR cells were exposed to pseudotyped viruses (Ada-M or HXB-2)
(250 ng of p24), Env-deficient viruses (250 ng of p24), or VSV-G
pseudotypes (0.2 ng of p24) at 37°C for 24 h. At this time point, TNF-␣ (10
ng/ml) (PeproTech) was added for 24 h at 37°C to induce virus gene expression (27). Finally, the cells were lysed, and luciferase activity was
monitored in each cell lysate as described previously (47). For experiment
aimed at blocking virus infection, the cells were pretreated for 30 min at
37°C with vehicle (used as a control), 0.1 ␮M bafilomycin A1, 10 mM
NH4Cl, or 1 mg/ml leupeptin (Sigma-Aldrich Canada). In these assays, the
drugs were present throughout the duration of the experiment. For esti-
Results
Trophoblastic cells are less susceptible to HIV-1 infection than
T lymphocytes
Trophoblasts form in vivo a polarized-like epithelium. Cell polarity is essential to the biological properties and functions of simple
epithelia because it leads to polarized endocytic pathways (apical
and basolateral), each of which uses a complex succession of intracellular compartments that include the early, late, and recycling
endosomes. These endocytic pathways control the fate of endocytosed macromolecules/pathogens. Hence, to study endocytic pathways in these cells, it is of utmost importance that cell polarity be
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The pNL4-3 (X4-tropic) (herein referred as to NL4-3 wild type) is a fulllength infectious molecular clone of HIV-1. The NL4-3 Luc⫹E⫺R⫹ vector
was constructed by inserting a frameshift mutation near the env gene and
the firefly luciferase-reported gene into the nef gene (supplied by N. R.
Landau, The Salk Institute for Biological Studies, La Jolla, CA) (44).
pHXB2-env is a mammalian expression vector coding for the HIV-1 HXB2
(X4-tropic) Env glycoprotein. This construct was obtained through the
AIDS Repository and Reagent Program. The pcDNA-1/Amp-based expression vector coding for the HIV-1 Ada-M (R5-tropic) full-length Env protein was generously provided by N. R. Landau. The pHCMV-G molecular
construct codes for the broad-host-range vesicular stomatitis virus Env glycoprotein G (VSV-G) under the control of the human CMV promoter (45).
Viruses were produced by calcium phosphate transfection of 293T cells as
described previously (27). Using the same protocol, the NL4-3 Luc⫹E⫺R⫹
expression vector was used to produce Env-deficient reporter viruses
(called NL4-3⌬Env).
Polarized JAR cells were pretreated with vehicle, 0.1 ␮M bafilomycin A1,
10 mM NH4Cl, or 10 nM wortmannin for 30 min at 37°C. The cells were
then exposed to viruses pseudotyped with VSV-G, Ada-M, or HXB-2 Env
(250 ng of p24) for 4 h (for 30 min and 2 h in some experiments) at 37°C.
At this point, the cells were washed three times with PBS and trypsinized
for 5 min at 37°C. The cells were pelleted by centrifugation (1200 rpm for
5 min at 4°C) and washed twice with ice-cold PBS. To disrupt cellular
membranes, cells were resuspended in 600 ␮l of ice-cold hypotonic buffer
(10 mM Tris-HCl (pH 7.5), 10 mM KCl, and 1 mM EDTA) for 1 min and
broken down by Dounce homogenization (three strokes, 7-ml B pestles).
Nuclei, cell debris, and undamaged cells were pelleted by centrifugation
(1800 rpm for 5 min at 4°C). Supernatants containing cytosol and vesicles
(including endosomes) were centrifuged at 12,000 rpm for 90 min at 4°C
in a Heraeus centrifuge. Supernatant that represents the cytosolic fraction
was adjusted to 0.5% Triton X-100, while the pellet, which is the vesicular
fraction, was resuspended in lysis buffer (20 mM HEPES (pH 7.4), 150
mM NaCl, and 0.5% Triton X-100). The level of p24 present in each
fraction was assessed using the p24 test.
Molecular constructs and preparation of virus stocks
6520
ENDOCYTOSIS OF HIV-1 IN HUMAN POLARIZED TROPHOBLASTS
viously reported real-time PCR test, which provides a measure of
true viral integration into the host cell chromatin. Indeed, this technical strategy uses primer sets designed to encompass both cellular
and HIV-1-specific DNA sequences (48). Briefly, a first round of
PCR amplification was conducted using Alu-specific (Alu) and
HIV-1-specific (M661) primers. This allows for amplification of a
region that encompasses both cellular and HIV-1-specific DNA
sequences and therefore amplifies only integrated viral DNA. The
first PCR product was subsequently subjected to a real-time PCR
assay targeting the HIV-1 R/U5 DNA region. This second PCR
step allows for quantification of the amount of integrated viral
DNA. The sum of these two PCRs is herein called integrated viral
DNA. It is important to state that because the second round of PCR
uses HIV-1-specific primers, it is necessary to control this experiment by conducting PCR on samples that have not undergone the
first round because retro-transcribed DNA would be amplified by
the second PCR. Samples that underwent only the second PCR are
herein referred to as “reverse-transcribed viral DNA.” High quantities of integrated HIV-1 DNA copies were detected in polarized
trophoblasts upon infection with wild-type NL4-3 virions (Table
I). Importantly, the number of viral DNA copies obtained upon
infection with NL4-3 was severely reduced upon treatment with
AZT (i.e., 66%), agreeing with the notion of true HIV-1 infection
in these cells. We next tested viral integration using PBMCs and
Jurkat cells. High quantities of integrated HIV-1 DNA copies were
also detected in both cell types, and as expected, such levels were
diminished by AZT. Interestingly, the number of integrated HIV-1
DNA copies was 2 logs higher in PBMCs and Jurkat cells compared with polarized JAR cells. Access to the cytoplasm, reverse
transcription (as controlled with AZT in LTR-reporter gene expression assay) and viral DNA integration are potent markers of
true HIV-1 infection in CD4⫹ T lymphocytes. Because all these
events are also observed in trophoblasts following exposure to
HIV-1 (this article and Ref. 27), it can be concluded that true virus
infection has indeed occurred in these cells.
Blocking the function of the endosomes inhibits HIV-1 infection
We have recently shown that the contact between HIV-1 and trophoblasts results in massive and rapid uptake of virions within
cells by endocytosis (27). Importantly, blocking the function of
endosomes in these cells inhibits infection with the R5-tropic
strain Ada-M, suggesting that events associated with HIV-1 infection in these cells are endocytic in nature (33). In the present work,
we extended this notion and studied infection with an X4-tropic
variant in the presence of two classes of endosome inhibitors, i.e.,
FIGURE 1. Susceptibility of polarized trophoblasts to HIV-1 infection. Polarized JAR cells (A) or Jurkat cells (B) were exposed to HXB-2 pseudotyped
reporter viruses for 24 h at 37°C. The cells were then stimulated for 24 h with TNF-␣ (10 ng/ml). Luciferase activity was monitored in each cell lysate.
Values from the luminometer are expressed as RLU. Data shown are expressed as the means ⫾ SD of quadruplicate samples and are representative of three
independent experiments.
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maintained. Purification of primary trophoblasts is extremely difficult, but more importantly, it results in the loss of cell polarity
(49). To resolve this issue, the human trophoblastic cell line JAR,
which exhibits many characteristics of the early placenta (50), was
used. Indeed, when grown on permeable supports, they form a
polarized epithelium-like monolayer closely resembling the trophoblastic barrier in vivo. This model system, which provides an
elegant alternative to primary cells (33), was used in this study.
To define the susceptibility to HIV-1 infection of polarized trophoblasts, comparative analyses were performed with a T lymphoid cell line (i.e., Jurkat) and unseparated PBMCs. First, polarized JAR cells were exposed to reporter viruses pseudotyped with
HXB-2 Env (X4-tropic). Although a weak luciferase activity was
detected following incubation with the two tested concentrations
of viruses in the absence of TNF-␣, this activity was not due to true
infection but was probably associated with the residual transfected
DNA and/or free-secreted luciferase protein present in our viral
stocks. Indeed, an inhibitor of reverse transcription, AZT, did not
reduce these observed relative light unit (RLU) values (Fig. 1A). In
contrast, luciferase activity could be readily detected in Jurkat cells
in the absence of TNF-␣. Indeed, a 24-fold increase in reporter
gene activity was seen when compared with mock-infected Jurkat
cells (Fig. 1B). We have demonstrated in a previous publication
that physiological concentrations of TNF-␣ are important for inducing HIV-1 long terminal repeat (LTR)-driven gene expression
in trophoblasts (27). Several cytokines, including TNF-␣, play pivotal roles during gestation and are mandatory for a successful pregnancy. It is postulated that the presence of TNF-␣ in our assays
parallels in vivo situations by creating conditions that lead to
higher HIV-1 expression and thus increased probabilities of vertical transmission of this retrovirus (27). In agreement with this
statement, when TNF-␣ was added to HXB-2-infected polarized
JAR cells, we observed a marked induction in LTR-driven gene
expression, reaching 28- and 46-fold increase following infection
with 125 and 250 ng of p24, respectively (Fig. 1A). Importantly,
AZT severely reduced this cytokine-induced LTR-driven gene expression, indicating that true infection had occurred in polarized
JAR cells (Fig. 1A). In these conditions, the induction triggered by
TNF-␣ was more potent in Jurkat cells where a 159-fold increase
in HIV-1 LTR-driven gene expression was detected (Fig. 1B).
Next, the presence of integrated viral DNA was assessed in polarized trophoblastic cells upon infection with wild-type NL4-3
viruses. JAR cells were either left untreated or exposed to wildtype NL4-3 viruses for 48 h, and total cellular DNA was isolated.
To quantify the amount of integrated viral DNA, we used a pre-
The Journal of Immunology
6521
Table I. Quantification of reverse transcribed and integrated HIV-1-specific DNAs following infection of
studied cells with fully competent NL4 –3 viruses
Integrated Viral DNAa
Cell Type
Reverse Transcribed
Viral DNAa
Without AZT
With AZT
Percentage of
Reduction
Polarized JAR
PBMCs
Jurkat
5 ⫾ 0.5
14.1 ⫾ 1.7
12.2 ⫾ 1.9
20 ⫾ 5.7
1340 ⫾ 78
3639 ⫾ 942
6.8 ⫾ 0.5
72 ⫾ 7
1684 ⫾ 152
66
95
54
a
Number of viral DNA copies per microliter (⫻103).
were pretreated with bafilomycin A1, virus gene expression was
virtually lost for all virus preparations tested (Fig. 2A). In regard to NH4Cl, although the effect was strong it was slightly
less potent than bafilomycin A1 (Fig. 2B). Collectively, these
data indicate that R5- and X4-tropic viruses are equally susceptible to the endosome inhibitors tested. Moreover, the process is independent of Env because infection with NL4-3⌬Env
viruses was also inhibited by bafilomycin A1. Because scientists have relied extensively on endosome inhibitors such as
bafilomycin A1 and NH4Cl to differentiate between pH-dependent (e.g., influenza virus and Semliki Forest virus) and pHindependent viral entry (reviewed in Ref. 53), it can be concluded that HIV-1 infection of trophoblasts is pH dependent.
FIGURE 2. Blocking the function of the endosomes inhibits HIV-1 infection. Polarized JAR cells were pretreated with bafilomycin A1 (A) or NH4Cl
(B) and exposed to pseudotyped and Env-deficient viruses for 24 h. The cells were next stimulated with TNF-␣. For kinetics of infection, polarized JAR
cells were exposed to HIV-1 particles for 6 h and next stimulated with TNF-␣ for 24 h. In such experiments, bafilomycin A1 (C) or NH4Cl (D) was either
omitted (0⬘) or added 30 min before virus infection (30⬘ pre) or 6, 12, and 24 h after HIV-1 infection. The cells were lysed, and luciferase activity was
monitored in each cell lysate. Values from the luminometer are expressed as RLU. Data shown are expressed as the means ⫾ SD of quadruplicate samples
and are representative of three independent experiments.
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the inhibitor of vacuolar proton ATPase bafilomycin A1 and the
lysosomotropic weak base NH4Cl. Both drugs block vesicle
acidification as well as endosomal and lysosomal degradation
systems (51, 52). Because, we have shown that HIV-1 infection
of trophoblasts occurs independently of the viral Env proteins
gp120 and gp41 (G. Vidricaire and M. J. Tremblay, submitted
for publication), Env-deficient HIV-1 particles were also tested
in this study. Polarized JAR cells were inoculated with singlecycle reporter viruses pseudotyped with Ada-M and HXB-2
(X4-tropic) Env or with Env-deficient reporter viruses (NL43⌬Env). Of note, a physiological concentration of TNF-␣ is
added in all our infection studies because its presence is important for inducing LTR-driven gene expression in trophoblasts and as such was used in these assays (27). When cells
6522
ENDOCYTOSIS OF HIV-1 IN HUMAN POLARIZED TROPHOBLASTS
Endosome inhibitors act at an early stage of HIV-1 replication
Endosome inhibitors vs HIV-1 internalization
We next verified whether the drugs had an impact on virus internalization. JAR cells were pretreated with bafilomycin A1 or
NH4Cl and then briefly exposed to pseudotyped or Env-deficient
viruses (i.e., 1 h) before an acid treatment to remove uninternalized
viruses. The level of the viral core protein p24 in the cell lysates
was quantified through the use of an enzymatic assay. Bafilomycin
A1 induced a 31, 43, and 55% reduction in internalization of
Ada-M, HXB-2, and NL4-3⌬Env viruses, respectively (Fig. 3A).
Bafilomycin A1 blocks not only vesicle acidification but also organelle movements from early to late endosomes (52). In the presence of bafilomycin A1, internalized molecules cannot proceed
beyond early endosomes and are thus returned to the extracellular
milieu. In such conditions, it is quite conceivable that bafilomycin
A1 triggers in part the recycling of HIV-1 to the apical pole, thus
creating the impression of reduced viral entry. Bafilomycin A1 was
also tested on HIV-1 binding to trophoblasts at 4°C and did not
modulate this event (data not shown). On the opposite, comparable
amounts of internalized viruses were found in cells treated or not
with NH4Cl and this for the three virus preparations tested
(Fig. 3B).
Endosome inhibitors induce a diminution in HIV-1 p24 cytosolic
release
Although a reduction in virus uptake is seen with bafilomycin A,
it is unlikely to be the sole reason for the marked decrease in
HIV-1 infection observed in polarized trophoblasts. Indeed,
NH4Cl, which also blocks infection, did not reduce significantly
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To determine the kinetics of HIV-1 travel inside the endosomal
machinery and of its eventual escape from acidic endosomal compartments, the time required for HIV-1 to become resistant to inhibition by bafilomycin A1 or NH4Cl was determined. Polarized
JAR cells were exposed to the studied drugs either before virus
infection or at various time points following it. In this experimental
setting, viral particles were added to the cells for 6 h at 37°C in the
absence of inhibitors to allow viral infection to proceed. At this
time point, infected cells were washed and treated with TNF-␣ to
promote viral gene expression. The endosome inhibitors were
added 6, 12, or 24 h after the onset of HIV-1 infection. As a
control, the cells were pretreated for 30 min with bafilomycin A1
or NH4Cl before infection. The determination of the time points
for adding the inhibitors to the cells was based on the following
criteria. First, for productive infection to be seen, polarized JAR
cells need to be in contact with HIV-1 for 6 h (our unpublished
observations). Second, adding the endosome inhibitors before infection and then only 6 h postinfection (i.e., once viral particles
have been washed off the cells) allows differentiating the effect of
the drugs on HIV-1 life cycle without impacting the process of
HIV-1 uptake. Treating polarized JAR cells with bafilomycin A1
30 min before virus infection yielded a 99% inhibition in viral
gene expression for Ada-M and HXB-2 pseudotypes and a 94%
reduction for Env-deficient viruses (Fig. 2C), which is in agreement with the results presented in Fig. 2, A and B. However, when
bafilomycin A1 was added 6, 12, or 24 h after initiation of virus
infection, the effect of the drug was gradually lost. Similar observations were made with NH4Cl (Fig. 2D), although this drug was
initially less potent than bafilomycin A1. Therefore, it would appear that both drugs act within the first 6 h of virus infection.
Hence, the requirement for low pH is associated with early events
of the HIV-1 life cycle. Interestingly, this timing coincides with
the presence of HIV-1 within the endosomal machinery (27, 33).
viral uptake. We next sought to define how blocking the endosome
translates into a reduction of HIV-1 infection. Because HIV-1
needs to access the cytoplasm for infection to ensue, cytosolic
release of the viral nucleocapsid p24 is believed to be an indicator
of productive infection (34). Consequently, drugs causing a reduction in cytosolic release of p24 might be associated with reduced
infection resulting from diminished endosomal escape. In other
words, we verified whether blocking the function of the endosomes
translates into diminished cytoplasmic release of viral material.
First, in a control experiment, polarized JAR cells were exposed to
HXB-2 pseudotypes for either 30 min or 2 h. The infected cells
were then trypsinized, washed, and the amount of viruses present
in the cytosolic and vesicular fractions was measured. We found
that at 30 min postinfection, the viral material was exclusively
located within the vesicular fraction of the cells, whereas at 2 h
postinfection, 20% of the internalized viral particles had already
reached the cytoplasm (Fig. 3C). These data are fully in agreement
with a previous report published by this laboratory on JAR cells
using this same technique (27) and underscore two important notions. First, the technical procedure used in this study for separating cytosolic and vesicular fractions is valid because it does not
result in cross-contamination of either fraction. Second, they illustrate clearly the endocytic nature of HIV-1 uptake in polarized
JAR cells. A second series of controls were also performed. It is
well established that entry of VSV occurs by receptor-mediated
endocytosis. Subsequently, during its itinerary through the endosomal compartments, the VSV-G protein acquires a low pH-induced fusion-competent form, allowing for fusion of the viral
membrane with endosomal and lysosomal membranes (54). Based
on this notion, polarized JAR cells were pretreated or not with
bafilomycin A1 or NH4Cl and then exposed for 2 h to NL4-3⌬Env
viruses pseudotyped with the VSV-G envelope, and fractionation
assays were conducted. Although VSV-G is internalized by endocytosis, we found that VSV-G viral particles were predominantly
located within the cytosolic fraction of polarized JAR cells. This
indicates that VSV-G efficiently reaches the cytoplasm upon internalization in trophoblasts in such a way that the duration of its
presence within the endosomes is short. These data are in agreement with a previous report (G. Vidricaire and M. J. Tremblay,
submitted for publication) and with the fact that VSV-G is highly
infectious in polarized JARs. However, upon treatment with
bafilomycin A1, no viral particles could escape the endosomes
because all viral particles were located within the vesicular fraction (Fig. 3D). This shows both the validity of the fractionation
procedure and the potency of the drug. Although NH4Cl reduced
the cytoplasmic presence of VSV-G pseudotypes from 83 to 56%,
the effect was less potent than that of bafilomycin A1. Hence, we
may conclude that NH4Cl is not as efficient as bafilomycin A1 for
blocking the cytoplasmic release. Next, fractionation assays were
performed in polarized JAR cells that were pretreated with bafilomycin A1 or NH4Cl and then exposed to Ada-M and HXB-2
pseudotyped viruses. The presence of bafilomycin A1 resulted in a
diminution of HIV-1 within the cytoplasm. Indeed, a shift from 36
to 23% ( p ⬍ 0.01) of p24 present in the cytoplasm was seen for
Ada-M, and a shift from 45 to 17% ( p ⬍ 0.01) was observed for
HXB-2 (Fig. 3, E and F, respectively). NH4Cl did not significantly
alter the cytosolic release of Ada-M or HXB-2. Given that NH4Cl
only partly modulated the cellular distribution of VSV-G
pseudotypes, it is not surprising that it did not modulate the cellular
distribution of HIV-1. We may conclude that bafilomycin A1, by
blocking the function of the endosomes, inhibits HIV-1 infection
of polarized JAR cells. The mechanism may be in part associated
with a reduced access to the cytoplasm.
The Journal of Immunology
6523
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FIGURE 3. Virus internalization and cytosolic delivery are reduced by bafilomycin A1. Polarized JAR cells were first pretreated or not with bafilomycin
A1 (A) or NH4Cl (B). The cells were then exposed to pseudotyped and Env-deficient viruses for 1 h at 37°C. Viral entry was assessed by evaluating the
amount of p24 in each cell lysate. Data shown are expressed as the means ⫾ SD of triplicate samples and are representative of three independent
experiments. For fractionation assays, polarized JAR cells were pretreated or not with bafilomycin A1 or NH4Cl and then exposed to HXB-2 (C and F),
VSV-G (D), and Ada-M (E) pseudotypes. The cells were disrupted by Dounce homogenization, and levels of p24 were evaluated in each cellular fraction.
Data shown are expressed as the means ⫾ SD of the ratio of p24 present in the cytosol or vesicles over the sum of p24 present in the two fractions. Data
shown are representative of three independent experiments. Asterisks denote statistically significant data (ⴱⴱ, p ⬍ 0.01). Statistical analysis was achieved
using the Student paired t test, with a two-tailed distribution. Homogeneity of variances was also tested and deemed as such in each case.
Rab5 regulates HIV-1 infection
In the next series of experiments, the role played by specific Rab
proteins in HIV-1 infection of polarized trophoblasts was studied.
The primary objective of these experiments was to define the endocytic compartment(s) involved in the process of HIV-1 infection
in this cell type. We recently provided evidence that HIV-1 is
internalized in trophoblasts via a clathrin/caveolae-independent
endocytic pathway that is sensitive to cholesterol-disrupting drugs
(G. Vidricaire and M. J. Tremblay, submitted for publication). Interestingly, additional results show that although HIV-1 is initially
6524
ENDOCYTOSIS OF HIV-1 IN HUMAN POLARIZED TROPHOBLASTS
the amount of DNA used for transfection and held true for the two
others virus preparations tested (Fig. 4, B and C). In contrast,
HIV-1 particles pseudotyped with VSV-G Env were weakly affected by the expression of the Rab5 proteins (Fig. 4D). We may
conclude that the effects observed by expression of the Rab5 mutants in trophoblasts are 1) specific to HIV-1, 2) related to early
events in HIV-1 replication, and 3) not related to downstream
events affecting HIV-1 LTR-driven gene expression. Hence, by
blocking access to early endosomes with dominant-negative Rab5
or conversely by preventing movements beyond early endosomes
with wild-type or dominant-active Rab5, virus infection is reduced
in polarized JAR cells. The effect cannot be due to decreased
HIV-1 internalization because similar amounts of viral particles
are found in polarized JAR cells expressing these Rab5 mutants
(see below).
Viral uptake and cytosolic p24 release are not affected by
wortmannin
Previous work has shown that inhibition of PI3K with wortmannin
leads to the release of EEA1 from early endosomes, which reduces
early endosome fusion (55). To better define the requirement for
FIGURE 4. Rab5 regulates HIV-1 infection. Polarized JAR cells were either mock-transfected or transfected with EGFP (control), Rab5 WT, Rab5
S34N, and Rab5 Q79L expression vectors. The cells were exposed to Ada-M pseudotypes (A), Env-deficient viruses (B), HXB-2 pseudotypes (C), or
VSV-G pseudotypes (D) and stimulated with TNF-␣. Luciferase activity was monitored in each cell lysate. Values from the luminometer are expressed as
RLU. Data shown are expressed as the means ⫾ SD of quadruplicate samples and are representative of three independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
localized in transferrin-negative organelles, internalized viral particles will eventually transit to transferrin-positive endosomes,
suggesting a merger between nonclassical and classical endocytic
pathways in trophoblasts (33). To define the relationship between
the presence of HIV-1 within early endosomes and the infection of
polarized trophoblasts, we investigated the role played by the small
GTPase Rab5, which regulates the biogenesis of these endosomes.
Polarized JAR cells were transiently transfected with expression
vectors coding for wild-type, dominant-active, and dominant-inactive Rab5. Substitution of glutamine to leucine at aa 79 (Rab5
Q79L) results in a Rab5 protein that cannot hydrolyze GTP, leaving it in an active state. In contrast, mutation of the serine to an
asparagine at position 34 (Rab5 S34N) causes the protein to have
an affinity for GDP that is 100-fold greater than that of GTP, therefore maintaining the protein in an inactive state. As controls, the
cells were either mock-transfected or transfected with the EGFP
expression vector. Contrary to the dominant-negative Rab5 (i.e.,
Rab5 S34N), the Rab5 WT and Rab5 Q79L molecular constructs
tested produced a potent but incomplete inhibition of infection
with Ada-M pseudotypes in polarized trophoblasts that ranged
from 50 to 76% (Fig. 4A). The effect observed was proportional to
The Journal of Immunology
HIV-1 to transit via Rab5⫹ organelles, the subsequent experiments
were performed with the PI3K inhibitor wortmannin. Although
wortmannin slightly increased Ada-M uptake in polarized JAR
cells, internalization of HXB-2 pseudotypes was not affected by
wortmannin (Fig. 5A). This is in agreement with the fact that internalization of HIV-1 does not proceed via the classical endocytic
pathway in trophoblasts (G. Vidricaire and M. J. Tremblay, submitted for publication). PI3K-dependent signaling pathway also
controls receptor sorting out of early endosomes but not membrane
transport (55). Keeping this in mind, the effect of wortmannin was
tested on cytosolic release of the viral nucleocapsid p24 in polarized JAR cells. Wortmannin did not modulate in a statistically
significant manner the cellular distribution of pseudotyped viruses
in polarized trophoblasts (Fig. 5B). Thus, it can be proposed that
HIV-1 cytoplasmic access from the endosomal machinery is most
likely independent of PI3K activity. In agreement with these results, wortmannin did not modulate HIV-1 infection of trophoblasts (data not shown).
Some transmembrane proteins internalized by a clathrin- and lipidraft-independent mechanism return to the plasma membrane in an
Arf6-dependent manner (Ras-related small GTP protein). Alternatively, they can fuse with the classical early endosomes and traffic
to late endosomes/lysosomes upon inactivation of Arf6. Expression of a constitutively active Arf6 mutant (Arf6 Q67L) prevents
molecules internalized in this clathrin/lipid raft-independent pathway to fuse with clathrin cargo-containing endosomes and their
subsequent routing to late endosomes. By contrast, exogenous expression of wild-type Arf6 has no discernible effect on cells (43).
Transient transfection of polarized JAR cells with wild-type Arf6,
dominant-active Arf6 Q67L, and dominant-negative Arf6 T27N
had no discernible effect on infection with HIV-1 pseudotyped
with Ada-M, HXB-2, or VSV-G Env (Fig. 6). Therefore, HIV-1 is
unlikely to transit via Arf6-expressing endosomes in trophoblasts.
Rab7 regulates HIV-1 infection
HIV-1 migrates and accumulates inside late endosomes within the
first hours of internalization into polarized trophoblasts (33). To
determine whether there is a correlation in polarized JAR cells
between the presence of HIV-1 within these organelles and the
process of infection, the effect of Rab7 was tested. The small GT-
Pase Rab7 controls movements of vesicles from early to late endosomes and the formation of lysosomes. Polarized JAR cells were
transiently transfected with dominant-negative and dominant-active Rab7 expression vectors. The two Rab7 proteins tested resulted in an almost complete blockade of HIV-1 gene expression in
trophoblasts. Indeed, up to 88% inhibition in viral expression was
observed (Fig. 7). The effect observed was proportional to the
amount of DNA used for transfection and held true for all three
strains of HIV-1 tested. In contrast, HIV-1 particles pseudotyped
with the VSV-G envelope were minimally affected by the expression of the Rab7 proteins (Fig. 7D). Hence, when blocking access
to late endosomes with dominant-negative Rab7 or conversely by
promoting movements to lysosomes with wild-type or dominantactive Rab7, HIV-1 infection is inhibited in polarized JAR cells.
The effect cannot be due to decreased HIV-1 internalization because comparable amounts of viral particles are found in polarized
JAR cells expressing these Rab7 mutants (Fig. 8). Similar data on
viral uptake were obtained when testing the Rab5 expression
vectors.
HIV-1 infection is independent of Rab11
Having shown in a previous study that a small proportion of internalized HIV-1 reaches Rab11-expressing recycling organelles
upon uptake into polarized trophoblastic cells (33), the possible
modulatory effect of different Rab11 constructs on HIV-1 infection
was tested (i.e., Rab11 WT, dominant-negative Rab11 S25N, and
dominant-active Rab11 Q70L). None of the Rab11 expression vectors tested had an effect on HIV-1 infection of polarized JAR cells
with reporter viruses (data not shown), thus suggesting that virus
infection of placental cells does not require a transit through these
organelles.
Discussion
The placenta is thought to play a central role during in utero HIV-1
transmission. However, the molecular events associated with
HIV-1 infection of placental cells are still a mystery. In the present
study, we investigated the role played by specific Rab proteins,
known to dictate the vesicular transport between the various endocytic compartments, in the process of HIV-1 infection of polarized human placental cells. The rationale of this study being that
HIV-1 circulates through multiple organelles upon internalization
into trophoblasts, and the presence of HIV-1 within the endosomes
FIGURE 5. Viral internalization and p24 cytosolic release are not affected by wortmannin. Polarized JAR cells were pretreated or not with wortmannin
and then exposed to Ada-M or HXB-2 pseudotyped viruses for 1 h (A). Viral entry was measured by evaluating the amount of p24 in each cell lysate. Data
shown are expressed as the means ⫾ SD of triplicate samples and are representative of three independent experiments. For fractionation assays, polarized
JAR cells were pretreated or not with wortmannin and then exposed to Ada-M or HXB-2 pseudotyped viruses for 4 h (B). The cells were disrupted by
Dounce homogenization, and levels of p24 were evaluated in each cellular fraction. Data shown are expressed as the means ⫾ SD of the ratio of p24 present
in the cytosol or vesicles over the sum of p24 present in the two fractions. Data shown are representative of three independent experiments.
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Arf6 does not modulate HIV-1 infection
6525
6526
ENDOCYTOSIS OF HIV-1 IN HUMAN POLARIZED TROPHOBLASTS
is a required step for infection to proceed in these cells. How both
events are interconnected is still unknown. We found an unprecedented relationship between placental infection and the presence
of HIV-1 within endosomes.
It is known that both HIV-1 fusion and endocytosis can occur
simultaneously in CD4⫹ T lymphocytes but with sharply different
functional consequences. Indeed, in this particular cell type, fusion
promotes productive infection whereas endocytosis leads to viral
degradation. Given that endosome inhibitors increase HIV-1 infectivity in CD4⫹ T cells and in an epithelial cell line (i.e., MAGI),
it was proposed that fusion within the endosomes is a possible
process when viral particles are spared from degradation (56, 57).
We found that the mechanism associated with HIV-1 infection of
polarized trophoblasts is utterly different. For all HIV-1 strains
tested, viral replication was not increased but rather virtually lost
when endosome inhibitors were added within the first moments of
infection. However, the endosome inhibitors did not reduce infection when added at a later time point following exposure to HIV-1,
most likely because a fraction of the virus had proceeded beyond
the low pH-sensitive step. We conclude that the initial presence of
HIV-1 within the endosomes in trophoblasts is mandatory for the
subsequent steps leading to infection. HIV-1 infection of trophoblasts, unlike CD4⫹ T cells, is thus endocytic in nature. Endocytosis of the retrovirus as a mechanism required for infection of a
target cell is rare and poorly characterized. Inhibition of macropinocytosis in macrophages leads to reduced viral infection. Although, most virions are subsequently degraded, fusion leading to
capsid release in the cytosol is postulated to take place inside ves-
icles. Infection of macrophages is weakly affected by treatment
with bafilomycin A1 (58). Human mannose receptor-mediated
HIV-1 infection of astrocytes was inhibited by bafilomycin A and
NH4Cl, thus suggesting that infection in these cells also results
from HIV-1 endocytosis (59).
The key question we asked in this article is how the presence of
HIV-1 within the endocytic compartments relates to infection.
Among other events, Rab5 regulates clathrin-mediated endocytosis
and fusion of coated pits with early endosomes (36). We found that
Rab5 did not modulate viral uptake into polarized trophoblasts.
This agrees with our earlier observation that internalization of
HIV-1 in these cells is independent of clathrin-coated pits (G. Vidricaire and M. J. Tremblay, submitted for publication). However,
we observed that the dominant-negative Rab5 mutant, which
blocks fusion of incoming vesicles to early endosomes, inhibited
HIV-1 infection in trophoblasts. Hence, Rab5-dependent transport
to early endosomes is a necessary step for HIV-1 infection in these
cells. Remarkably, these observations also imply that HIV-1-containing endocytic vesicles derived from a Rab5-independent process are able to fuse with early endosomes that are presumably
Rab5 positive. Indeed, HIV-1 transits from the nonclassical to the
classical pathway following uptake by trophoblasts via a clathrin/
caveolae-independent pathway (G. Vidricaire and M. J. Tremblay,
submitted for publication). This constitutes an unusual and poorly
characterized event; one elegant example includes caveolar vesicles moving from caveosomes to early endosomes in a Rab5-dependent manner (35). Of note, the reason why Ada-M pseudotypes
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FIGURE 6. HIV-1 infection is independent of Arf6. Polarized JAR cells were either mock-transfected or transfected with EGFP (control), Arf6 WT,
Arf6 T27N, and Arf6 Q67L expression vectors. Next, the cells were exposed to Ada-M (A), HXB-2 (B), or VSV-G (C) pseudotyped viruses and stimulated
with TNF-␣. Luciferase activity was monitored in each cell lysate. Values from the luminometer are expressed as RLU. Data shown are expressed as the
means ⫾ SD of quadruplicate samples and are representative of three independent experiments.
The Journal of Immunology
6527
were not affected by the dominant-negative Rab5 vector is intriguing. Given that the difference between Ada-M and HXB-2 relates
to Env and that infection of trophoblasts is Env independent, this
point is difficult to interpret. It is possible that posttranslation modifications of Env (e.g., glycosylation pattern) differ between
Ada-M and HXB-2. These differences, although not impacting on
the early steps in HIV-1 life cycle, might modulate the fate of the
viral material within endocytic compartments. We also found that
wild-type and constitutively active Rab5 proteins inhibited HIV-1
infection. Dominant-active Rab5 Q79L causes the accumulation
within endosomes of lysosomal proteins that normally cross the
endosome compartments before reaching lysosomes (60). Likewise, small and large dextran molecules remain colocalized after
endocytosis in Rab5 Q79L-expressing cells, and their separation
into distinct endocytic organelles is delayed severely (61). Hence,
by causing entrapment of HIV-1 within early endosomes, Rab5
Q79L or Rab5 WT was detrimental to HIV-1 infection in trophoblasts. We conclude that although reaching early endosomes is
required for the biology of HIV-1 within trophoblasts, this step is
not sufficient per se for infection to take place.
Importantly, we found that HIV-1 infection was virtually lost
upon expression of a dominant-negative Rab7 mutant, which
blocks the transport to late endosomes and results in retention in
Rab5-positive early endosomes. Because dominant-active Rab5
and dominant-negative Rab7, which both prevent access to late
endosomes, inhibited infection, we can conclude that HIV-1 trafficking to late endosomes is a mandatory step for infection to proceed in trophoblasts. This also implies that infection is related to
events not occurring within early endosomes but rather within late
endosomes. In this context, the role of Rab5 is to promote HIV-1
transit to early endosomes, a step known to be important for subsequently reaching late endosomes via Rab7. Of note, expression
of Rab5 expression vectors in polarized trophoblasts reduced
HIV-1 infectivity by 50 to 76%, whereas the observed Rab7-mediated inhibition was much more dramatic. Transfection efficiencies obtained in this study (i.e., 50%) parallel the inhibition
achieved by the Rab5 expression vectors and as such may explain
the extent of inhibition obtained with these vectors. This being
said, this argument is difficult to make because it would imply that
a direct correlation exists between transfection efficiency, viral uptake, and infection in trophoblasts. However, a direct association
between viral uptake and infection is unlikely to be the case in
these cells. Indeed, even though virtually 100% of polarized JAR
cells internalize HIV-1 viral particles (33), infection rates are expected to be much lower in these cells. This is based on the idea
that a maximum of 10% of CD4⫹ T cells are infected by HIV-1
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FIGURE 7. Rab7 regulates HIV-1 infection. Polarized JAR cells were either mock-transfected or transfected with EGFP (control), Rab7 T22N, and Rab7
Q67L expression vectors. Next, the cells were exposed to HXB-2 pseudotypes (A), Ada-M pseudotypes (B), Env-deficient viruses (C), or VSV-G
pseudotypes (D) and stimulated with TNF-␣. Luciferase activity was monitored in each cell lysate. Values from the luminometer are expressed as RLU.
Data shown are expressed as the means ⫾ SD of quadruplicate samples and are representative of three independent experiments.
6528
ENDOCYTOSIS OF HIV-1 IN HUMAN POLARIZED TROPHOBLASTS
both in vivo and in vitro (our unpublished observations and Ref.
62) and that trophoblasts are recognized to be even less susceptible
to HIV-1 infection. In other words, all trophoblastic cells internalize HIV-1 but only a subset of them becomes infected. Hence, our
data raise the alternative and intriguing possibility that HIV-1 particles reach late endosomes not solely via Rab5-positive early endosomes but possibly via yet to be defined Rab5-negative early
endosomes as well.
In contrast, we provide evidence that infection is unlikely to be
associated with a transit via Arf6- or Rab11-containing organelles
given that Arf6 and Rab11 mutants did not modulate HIV-1 infection in polarized trophoblasts. It has been demonstrated previously that the bulk transport from early to late endosomes is not
affected after inhibition of the PI3K pathway by wortmannin but
that the sorting of down-regulated receptor molecules out of early
endosomes is inhibited (55). In apparent contradiction with a mandatory transit to late endosomes for HIV-1, we observed that wortmannin failed to inhibit HIV-1 cytoplasmic access in polarized
trophoblasts. Consequently, HIV-1 particles reach late endosomes
in a process resembling bulk transport and as such do not require
PI3K. This is supported by our earlier finding that uptake of HIV-1
in trophoblasts occurs via numerous and rather nonspecific viruscell interactions (G. Vidricaire and M. J. Tremblay, submitted for
publication).
It is important to point that our data also illustrate the existence
of a delicate balance between the organelles. Indeed, if movements
from late endosomes toward lysosomes are favored upon expression of dominant-active Rab7, HIV-1 infection is inhibited. This
may be due to increased viral degradation within lysosomes based
on the idea that Rab7 Q67L increases perinuclear aggregation and
fusion of lysosomes. It is surprising that infection by HIV-1
pseudotyped with VSV-G was poorly modulated by a dominantnegative Rab5 mutant as described previously (63). The difference
in cell lines may account for such a discrepancy. It is unlikely to
be due to VSV-G infection rates that may not be overcome by the
levels of expression achieved by Rab expression vectors. Indeed,
when performing these experiments with a titer of VSV-G
pseudotypes giving more comparable luciferase readouts to HIV-1
pseudotypes, VSV-G remained unaffected (data not shown).
Internalization of viruses within a given target cell is in itself
generally not sufficient per se for productive infection because incoming viruses are still part of the extracellular space once located
within endosomes. The acidification of endocytic vesicles triggers
a conformational change in the viral Env protein of enveloped
viruses causing fusion of the virus with the endocytic inner membrane and the release of viral genome at appropriate locations (reviewed in Ref. 64). In contrast, naked viruses are released upon
rupture of the cargo endocytic vesicles; alternatively, they generate
a pore through which the genome can enter the cytosol (reviewed
in Refs. 64 and 65). In trophoblastic cells, the cytosolic p24 content decreased in the presence of bafilomycin A1, suggesting that
when endosomal function is blocked, the mechanism whereby viral particles present in vesicles are released into the cytosol is
inhibited and as such infection is lost. Hence, these data support
the notion that HIV-1 infection in polarized trophoblasts is associated with a pH-dependent endosomal escape. If a strict correlation between the presence of viral material within the cytoplasm
and infection exists in trophoblasts as it is the case in CD4⫹ T
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 8. HIV-1 uptake is not modulated by Rab5 and Rab7. Polarized JAR cells were either mock-transfected or transfected with EGFP (control),
Rab5 WT, Rab5 S34N, Rab5 Q79L, Rab7 T22N, and Rab7 QP67L expression vectors. Next, the cells were exposed to Ada-M (A and C) or HXB-2 (B
and D) pseudotyped viruses for 1 h. Viral entry was measured by evaluating the amount of p24 in each cell lysate. Data shown are expressed as the means ⫾
SD of triplicate samples and are representative of three independent experiments.
The Journal of Immunology
In summary, this study provides an unprecedented understanding of the intracellular route taken by HIV-1 within human placental cells. The results presented herein are highly significant.
First, the mechanism underscored in this work has never been described for HIV-1. Second, the human polarized trophoblast barrier is considered as a primary target for maternal blood-borne
HIV-1 infection. Third, the results obtained can also enhance our
understanding of the biology of HIV-1 in adults. Indeed, epithelial
cells are the portal of entry for HIV-1 during primary infection in
adults, and viral internalization is associated with endocytosis in
these cells. Moreover, endocytosis may be important for macrophages and dendritic cells, which are cell types playing key roles
in the pathogenesis of HIV-1 infection. Furthermore, the findings
presented in this article raise concerns in terms of treatment strategy. Administration of clinically approved drugs such as amantadine or chloroquine has been postulated to increase HIV-1 infectivity by sparing viruses from degradation in late endosomes and
lysosomes (56, 57). Based on the present data, it can be proposed
that an opposite effect would be seen with these drugs with respect
to vertical transmission of HIV-1. In contrast, our data raise the
interesting possibility that altering the endocytic Rab content by
intervention in appropriate signal transduction events may offer a
new strategy for modulating the targeting of HIV-1 to a degradative compartment (69). Additional studies are needed to define the
validity of this approach.
Disclosures
The authors have no financial conflict of interest.
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cells, it is intriguing that the cytosolic p24 content was reduced and
not blocked by bafilomycin A1 and was not affected by NH4Cl.
Controls were performed to ensure that the technical procedure to
separate cytosolic and vesicular fractions was proper; hence, it is
unlikely due to cross-contamination between the fractions. In contrast, the fact that the cytosolic release of VSV-G was completely
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blockade in cytoplasmic access triggered by the drugs used is related to the nature of the assay. This being said, both drugs blocked
viral infection within the first 6 h of infection, implying that they
abrogate steps within the early events of HIV-1 infectious cycle.
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Among other events, low endosomal pH regulates activation of
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found that endosome inhibitors blocked infection of polarized trophoblasts with wild-type and Env-deficient viruses. Therefore, we
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independent of the Env proteins. Pivotal information is now to be
considered. First, HIV-1 is primarily located within late endosomes upon internalization in trophoblasts (33). Second, inhibiting
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