From the Centre de Recherche en Infectiologie, Hôpital CHUL, Centre Hospitalier Universitaire de Québec, and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec G1V 4G2, Canada
Received for publication, October 11, 2002, and in revised form, February 6, 2003
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ABSTRACT |
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Maternal-infant transmission of human
immunodeficiency virus type-1 (HIV-1) is the primary cause of this
retrovirus infection in neonates. Trophoblasts have been proposed to
play a critical role in modulating virus spread to the fetus. This
paper addresses the mechanism of HIV-1 biology in trophoblastic cells.
The trophoblastic cell lines BeWo, JAR, and JEG-3 were infected with
reporter HIV-1 particles pseudotyped with envelope glycoproteins from
the vesicular stomatitis virus or various strains of HIV-1. We
demonstrate that despite a high internalization process of HIV-1 and no
block in viral production, HIV-1 established a limited infection of
trophoblasts with the production of very few progeny viruses. The
factor responsible for this restriction to virus replication in such a
cellular microenvironment is that the intracellular p24 is concentrated
predominantly in endosomal vesicles following HIV-1 entry. HIV-1
transcription and virus production of infectious particles were both
augmented upon treatment of trophoblasts with tumor necrosis factor- Mother-to-child transmission of the human immunodeficiency virus
type 1 (HIV-1)1 is a serious
public health issue. An estimated 2.4 million infected women give birth
annually, and 1,600 infants acquire HIV-1 infection every day worldwide
(reviewed in Ref. 1). In the absence of antiretroviral treatment, the
reported risk of vertical transmission lies within the range of
10-39% (1-3). Vertical transmission of HIV-1 from mother-to-child
can occur prepartum (in utero involving transplacental
passage) and intrapartum (at birth upon exposure of the skin and mucous
membranes of the infants to maternal blood and vaginal secretions)
(4-6). Mathematical modeling has allowed the estimation that 30% of
infections occur in utero less than 2 months before birth
and 65% at birth (7). Transmission of HIV-1 during early gestation
also occurs since HIV-1 has been detected in 8-week aborted fetuses and
in second trimester fetal tissues (8-11).
In order for the virus to reach the fetal circulation and infect the
fetus in utero, HIV-1 must cross the placental barrier which
is made of a double layer of polarized epithelial type cells, the
cytotrophoblasts and syncitiotrophoblasts. These cells separate the
maternal and fetal blood circulations and control fluxes between the
two circulations. Several published reports have addressed the question
as to how HIV-1 reaches the fetus. It has been shown that the placenta
may allow transcytosis of the virus from the maternal to the fetal
circulation (12). Alternatively and/or concomitantly, the virus may
infect the placenta to reach the fetal circulation and ultimately the
fetus. Indeed, HIV-1 has been detected on both the maternal and fetal
portions of the placenta, i.e. in decidual macrophages,
leukocytes, trophoblasts, Hofbauer cells, in villous endothelial cells,
and CD3-expressing placental cells (8, 13-18). Moreover, some
authors have shown that HIV-1 undergoes productive replication in
the placenta. The analysis of the evolutionary relationships of the
sequences of HIV-1 clearly linked maternal sequences with associated
sequences in the trophoblasts and put them on distinctive branches (3).
In addition, human choriocarcinoma cell lines (i.e. BeWo,
JAR, and JEG-3) as well as isolated primary trophoblastic cells
(including cytotrophoblasts from term placenta and cytotrophoblasts
from term placenta induced to differentiate in syncytiotrophoblasts
in vitro) and Hofbauer cells were shown to be weakly
permissive to HIV-1 infection in vitro and to sustain a low
level of virus replication. However, other scientists have failed to
detect the presence of HIV-1 in the placenta of infected mothers and/or
replication of the virus in these cells (19). Thus, it remains
controversial whether HIV-1 can sustain a productive cycle in
trophoblastic cells, but if indeed possible, it is clear that it is
magnitudes lower than in CD4-positive T lymphocytes. The reasons behind
this limited infection are ill defined but are thought to be due to
factors related to the phenotype of HIV-1 in conjunction with
particular cellular environments. This may include limitations in virus
entry, intracellular restriction, inappropriate cellular environment, or a combination of the three (reviewed in Ref. 20). The question of
viral entry is crucial because the expression of the cellular receptor
CD4 of HIV-1 is very low, whereas the expression of co-receptors, CXCR4
and CCR5, of HIV-1 may decline from the first to third trimester of
gestation. This may account for a limited viral entry (9, 21-24).
However, the process of viral entry per se in trophoblastic cells has not been investigated thus far.
A key feature of pregnancy is the production of a vast array of
cytokines (e.g. IL-1, IL-3, IL-4, IL-6, IL-10,
granulocyte macrophage-colony stimulating factor,
macrophage-colony stimulating factor, leukemia inhibitory factor,
transforming growth factor- The central objective of the present work was to provide further
insight on the susceptibility of human trophoblasts to a productive
infection with HIV-1. To this end, we conducted experiments with both
fully infectious HIV-1 particles and recombinant HIV-1-based reporter
viruses pseudotyped with the envelope proteins of the broad-host-range
vesicular stomatitis virus (VSV-G) and certain strains of HIV-1. The
pseudotyping strategy with VSV-G allows bypassing the natural mode of
HIV-1 entry and not only broadens the natural virus tropism but also
significantly enhances virus infectivity (30). Contrary to previous
assumptions, we demonstrate for the first time that the reason for the
limited HIV-1 infection of trophoblastic cells is not linked with
ineffective virus internalization because we found massive HIV-1 entry
in trophoblastic cells. Rather, the subcellular distribution of viral
p24 was predominantly located in the vesicular fraction, an event
accounting for the weak virus production in such cells. On the other
hand, we showed that trophoblastic cells possess no block in viral
transcription or viral production. We showed that TNF- Cells--
The malignantly transformed human cell lines of
trophoblast lineage BeWo, JAR, and JEG-3 were obtained from the
American Tissue Culture Collection (Manassas, VA). The human embryonic
kidney cell line 293T (expressing the simian virus 40 large T antigen) was kindly provided by W. C. Greene (J. Gladstone
Institutes, San Francisco). The JAR, JEG-3, and 293T cells lines
were cultured in DMEM (Invitrogen), and the BeWo cell line was
maintained in F-12 Nutrient Mixture (Invitrogen). Both media were
supplemented with 10% fetal bovine serum (FBS) (Invitrogen), glutamine
(2 mM), penicillin G (100 units/ml), and streptomycin (100 mg/ml). BeWo, JAR, and JEG-3 cells were routinely subcultured at a
seeding density of 3 × 106 cells and 293T cells at
1 × 106 cells in 75-cm2 tissue culture
flasks. PM1 cells were obtained through the AIDS Research and Reference
Reagent Program (Division of AIDS, NIAID, National Institutes of
Health, Bethesda). These cells were cultured in RPMI 1640 medium
supplemented with 10% FBS. The reporter LuSIV cell line, kindly
provided by J. E. Clements (The Johns Hopkins University
School of Medicine, Baltimore, MD), was derived from the CEMx174
parental cell line (B-cell/T-cell hybrid) and carries the luciferase
reporter gene under the control of the SIVmac239 LTR. These cells were
maintained at 0.5 × 106 cells/ml in selection medium
made of RPMI 1640 (Invitrogen) with 10% FBS, 15 mM NaOH,
25 mM HEPES, and supplemented with glutamine (2 mM), penicillin G (100 units/ml), streptomycin (100 mg/ml), and 300 µg/ml hygromycin B (Roche Applied Science). Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by
centrifugation through a Ficoll-Paque density gradient. PBMCs were
resuspended at a density of 106 cells/ml in RPMI 1640 with
20% FBS, 15 mM NaOH, 25 mM HEPES and supplemented with glutamine (2 mM), penicillin G (100 units/ml), streptomycin (100 mg/ml), 3 µg/ml of PHA-P (Sigma), and 50 units/ml of human recombinant IL-2 (kind gift of M. Gately,
Hoffmann-La Roche Molecular Biochemicals) (31).
Molecular Constructs--
pNL4-3 is a full-length infectious
molecular clone of HIV-1 and the NL4-3-Luc
E Preparation of Virus Stocks--
Viruses were produced by
calcium phosphate transfection of 293T cells, as described previously
(37, 38). Briefly, 293T cells were plated 16 h before transfection
to reach a 50-80% confluence the day of transfection. By using the
Clontech Transfection Kit (BD Biosciences), cells
were co-transfected with NL4-3-Luc E Virus Entry and Cell Fractionation Assays--
Approximately
1 × 106 of JAR and PM1 cells were incubated with
Ada-M or VSV-G pseudotyped HIV-1 particles (200 ng of p24) in 6-well
tissue culture plates at 37 °C for a period of 5 min to 4 h.
Cells were next extensively washed with ice-cold PBS and trypsinized
for 5 min at 37 °C. The cells were then washed with RPMI
supplemented with 10% FBS followed by three washes with ice-cold PBS.
For cell entry assays, cells were resuspended in lysis buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% Triton
X-100). The level of p24 was determined by an in-house enzymatic assay
as described previously (39). For cell fractionation assays, to disrupt
cellular membranes, cells were resuspended in 1 ml of ice-cold
hypotonic buffer (10 mM Tris-HCl (pH 7.5), 10 mM KCl, 1 mM EDTA) for 1 min and broken by
Dounce homogenization (three strokes, 7-ml B pestles). Nuclei, cell
debris, and undamaged cells were pelleted by centrifugation (1,800 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 1 ml of lysis buffer
(20 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% Triton X-100). The level of p24 present in each fraction was assessed using the p24 assay (39).
Virus Infection, Luciferase Assays, and Co-culture
Experiments--
In 25- or 75-cm2 tissue culture flasks,
2-6 × 106 BeWo, JAR, or JEG-3 cells were incubated
at 37 °C under a 5% CO2 atmosphere for 7 h with
luciferase-encoding HIV-1 particles pseudotyped with VSV-G (6-145 ng
of p24), Ada-M, or HXB2 envelope glycoproteins (250-400 ng of p24).
Cells were then washed three times with PBS, trypsinized, and
subcultured at 25 × 103 cells per well in 96-well
flat-bottom tissue culture plates or at 50 × 103
cells per well in 48-well flat-bottom tissue culture plates in 200 µl
of complete DMEM culture medium. After an overnight incubation, 100 µl of medium was removed from each well and replaced with 100 µl of
culture medium supplemented with the following agents: tumor necrosis
factor (TNF)- Transient Transfection of JAR Cells--
JAR cells were
transiently transfected by calcium phosphate precipitation using the
Clontech Transfection Kit. Briefly, 0.5-1.5 × 106 JAR cells were plated 16 h before transfection
in 25-cm2 tissue culture dishes. For each transfection, 5 µg of plasmid DNA was used. For the HIV-1 clades A to G transfection
experiment, JAR cells were co-transfected with 1 µg of an actin
promoter-driven Early Events in the HIV-1 Replicative Cycle Represent Rate-limiting
Steps for Virus Infection of Trophoblast-derived Malignant Cell
Lines--
Given the reported low susceptibility of trophoblastic
cells to productive HIV-1 infection (22, 23, 41), we initially defined
the permissiveness of malignantly transformed cell lines of trophoblast
lineage to the early events in HIV-1 biology. This goal was reached by
inoculating BeWo, JAR, and JEG-3 with luciferase expressing
single-cycle HIV-1 particles pseudotyped either with the envelope
protein of HXB2 (T-tropic HIV-1 strain), Ada-M (macrophage-tropic HIV-1
strain), or the envelope G protein of VSV (i.e. VSV-G). A
previous study (30) has shown that pseudotyping of HIV-1 particles with
VSV-G results in both a marked enhancement of virus infectivity and in
an extended cellular tropism. Exposure of BeWo, JAR, and JEG-3 cell
lines to HXB2 and Ada-M pseudotypes yielded a very low spontaneous
HIV-1 LTR-directed reporter gene activity (Table I). However, when these cells were
exposed to single-cycle VSV-G pseudotyped reporter viruses, a
significant enhancement in luciferase activity was observed. The
highest augmentation in HIV-1 transcriptional activity was obtained for
JAR and the lowest for BeWo. Based on these observations, it can be
concluded that the failure of HIV-1 to productively infect human
trophoblast cells is related to a blockade in the early steps in the
virus life cycle.
Virus Internalization Shows No Restriction in Trophoblastic
Cells--
We next verified whether the limitation in HIV-1 production
was at the level of internalization because trophoblastic cells are
reported to express either no or very little CD4 and CXCR4 or CCR5. JAR
cells were exposed to VSV-G pseudotypes, Ada-M pseudotypes, or fully
infectious NL4-3 for 5 min to 4 h at 37 °C. Subsequently, the
cells were trypsinized, washed, and lysed before monitoring the amount
of internalized virus. We found that the three virus preparations
tested were able to enter with high efficiency and in a timely fashion
in the trophoblastic cell line JAR (Fig.
1). Surprisingly enough, VSV-G and Ada-M
pseudotypes were found to enter JAR cells at a comparable level;
therefore suggesting that the blockade in HIV-1 transcriptional
activity in trophoblast cells is not associated with a barrier that
limits the process of HIV-1 attachment and internalization.
HIV-1 Is Found Predominantly in the Vesicular Fraction upon Viral
Entry into Trophoblastic Cells--
In order to define the exact
location of HIV-1 upon virus entry into trophoblasts, JAR cells were
exposed to Ada-M pseudotyped HIV-1 particles for a short time, and we
measured the amount of viruses present in the cytosolic and vesicular
fractions. A similar technical approach was used with PM1 cells, a
CD4-expressing T lymphoid cell line known to be highly susceptible to
HIV-1 infection. Although we observed comparable absolute amounts of
intracellular p24 levels in JAR and PM1 cells, differences were
detected with respect to intracellular p24 in cytosolic and vesicular
fractions. In JAR cells, at 30 min following virus exposure, Ada-M
pseudotyped viruses were present in the vesicular fraction only (Fig.
2A). Vesicular and cytosolic
p24 represented 82 and 18%, respectively, of total intracellular p24
after 2 h of virus exposure. The distribution of intracellular p24
between the cytosolic and vesicular fractions was different in PM1
cells. For example, after 2 h of exposure to Ada-M pseudotypes,
vesicular and cytosolic p24 in PM1 cells represented 65 and 35%,
respectively, of the total intracellular p24 (Fig. 2B). Our
findings indicate that HIV-1 internalization in trophoblastic cells is
a highly efficient process, but the route of entry is mediated in large
part through endosomal vesicles. Given that productive HIV-1 infection
has been proposed to result from the cytosolic release of p24 (42), the
reduced susceptibility of trophoblast to HIV-1 infection is due to an
extensive vesicular uptake.
HIV-1 LTR-driven Reporter Gene Activity Is Increased in
Trophoblastic Cells by TNF- Production of Fully Infectious HIV-1 Particles by Trophoblastic
Cells Is Only Seen Following Treatment with TNF- The Major Limiting Step of HIV-1 Infection in Trophoblasts Is at
the Level of the Route of Entry and Is Not Related to Intracellular
Restrictions--
The natural low susceptibility of trophoblast cells
to be productively infected with HIV-1 could be due, in addition to the demonstrated significant vesicular uptake, to some undefined
intracellular restrictions to virus replication. To shed light on this
issue, JAR cells were infected with fully competent
HIV-1NL4-3 viruses that bear also the VSV-G envelope
glycoproteins before exposure to TNF- Different HIV-1 LTR Subtypes Are Transactivated by
TNF- Cytokine-dependent Up-regulation of HIV-1 LTR Activity
in Trophoblasts Is Mediated via NF- The overall objectives of the present study was to provide
additional information on the possible mechanism(s) responsible for the
weak susceptibility of trophoblastic cells to productive HIV-1
infection and to define if cytokines present in the environment surrounding HIV-1-infected trophoblasts could positively modulate virus
expression. In the present report, a strong spontaneous activation of
the HIV-1 LTR domain was seen in the three choriocarcinoma cell lines
tested (BeWo, JAR, and JEG-3) following infection with HIV-1 particles
pseudotyped with VSV-G. This set of data clearly indicates that virus
transcription is highly efficient in trophoblastic cells. These results
are in agreement with a previous work that showed that choriocarcinoma
cell lines support basal and Tat-transactivated transcriptional
activity of HIV-1 reporter gene constructs (17). However, a minimal
HIV-1 LTR-mediated reporter gene activity was detected when infection
of trophoblastic cells was allowed to proceed with viruses bearing
macrophage-tropic and T-tropic HIV-1 envelope glycoproteins. This
series of investigations suggest that the most proximal events in HIV-1
biology represent major limiting steps of virus infection in trophoblasts.
It has been proposed that the reason behind the limited HIV-1 infection
of trophoblastic cells is due to a block at virus entry, which could be
caused by a minimal expression of CD4 and appropriate chemokine
co-receptor (21-24). In the present study, contrary to what was
assumed, we provide evidence that HIV-1 enters massively into
trophoblastic cells. Subcellular fractionation techniques that
segregate between vesicular and cytosolic fractions revealed that a
major part of intracellular p24 is found in endosomal vesicles,
implying thus that HIV-1 enters trophoblastic cells predominantly via
endocytosis. This route of entry reveals important consequences for the
virus life cycle in trophoblastic cells. Indeed, recent findings have
shown that HIV-1 entry can occur through plasma membrane fusion and
endocytosis in both HeLa and Jurkat cells. The latter mode of entry
usually leads to a nonproductive process of infection because viruses
will ultimately be degraded in lysosomes (43). In trophoblastic cells,
phagocytosis is thought to play an important role in the extensive
tissue remodeling that occurs during trophoblastic invasion of the
decidua and in the control of exchanges between the maternal and fetal
circulations. Moreover, trophoblastic cells actively transcytose
several molecules including immunoglobulins and even pathogens such as
HIV-1 (12). Thus, based on our findings and the current literature, it
is likely that a fraction of HIV-1 entering trophoblasts is
transcytosed and another part is trapped in endocytic vesicles and
ultimately degraded. The sum of these two mechanisms would account for
the low permissiveness of trophoblasts to productive HIV-1 infection.
The pro-inflammatory cytokines TNF- Concerning possible restrictions at late events in the HIV-1 life
cycle, we found that trophoblast cells sustained high virus production
when infection was performed with VSV-G pseudotypes HIV-1-based
viruses. Thus, it can be concluded that, apart from an inefficient
route of virus entry, there is no blockade at late steps in the
replicative cycle of HIV-1 in trophoblast. The minor fraction of
intracellular p24 that is found in trophoblast is not sufficient to
lead to a measurable virus production because a co-culture step with
indicator cells is necessary to detect virus production. Under in
vivo situations, trophoblasts are in close contact with cells such
as lymphocytes that show a higher susceptibility to HIV-1 entry and
productive infection. Thus, it can be proposed that despite an
inefficient mode of HIV-1 entry in trophoblasts, spreading of the virus
to the fetus via infection of trophoblasts is a possible scenario.
Indeed, viral production in trophoblasts could be triggered under
natural conditions upon exposure to TNF- In line with our results, the relative importance of TNF- TNF- The roles played by TNF- Taken together, the data presented in this report provide important
novel pieces of information in regard to the possible mechanism of
in utero transmission of HIV-1. We have shown that HIV-1
enters massively into trophoblastic cells, and data from infection
studies with VSV-G pseudotypes HIV-1 particles suggest that there are
no other intracellular restrictions in this cell type. The natural low
susceptibility of trophoblast cells to a productive HIV-1 infection is
linked with a block at the initial stage(s) of the virus life cycle and
more precisely with a vesicular uptake of p24, a process that leads to
degradation by lysosomal enzymes and/or transcytosis of the virus. We
further show that these limitations can be partially overcome by a
treatment with pro-inflammatory cytokines such as TNF-
and interleukin-1. However, the amount of progeny virions released by
trophoblasts infected with native HIV-1 virions was so low even in the
presence of pro-inflammatory cytokines that a co-culture step with
indicator cells was necessary to detect virus production. Collectively
these data illustrate for the first time that the natural low
permissiveness of trophoblasts to productive HIV-1 infection is because
of a restriction in the mode of entry, and such a limitation can be
overcome with physiologic doses of tumor necrosis factor-
and
interleukin-1, which are both expressed by the placenta, in conjunction
with cell-cell contact. Considering that there is a linear correlation
between viral load and HIV-1 vertical transmission, the environment may
thus contribute to the propagation of HIV-1 across the placenta.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, interferon-
and TNF-
), hormones,
growth factors (e.g. epidermal growth factor, vascular
endothelial growth factor, progesterone, and estrogen), and
prostaglandins (e.g. prostaglandin E2) in a developmentally regulated fashion by the conceptus and/or the uterus.
These agents play pivotal roles during gestation and are mandatory for
a successful pregnancy (reviewed in Refs. 25-27). On the other hand,
some of these agents are also known to be up-regulated in
vivo in HIV-1-infected patients and to modulate viral expression in vitro (reviewed in Refs. 20 and 28). We previously tested the ability of factors known to be present in the vicinity of the
trophoblast during gestation and for which trophoblastic cells express
the appropriate receptors to drive HIV-1 transcriptional activity in
trophoblasts. Among all the soluble agents tested (i.e.
epidermal growth factor, granulocyte macrophage-colony
stimulating factor, interferon-
, IL-1
, IL-1
, IL-3, IL-4, IL-6,
IL-7, IL-8, IL-10 IL-12, leukemia inhibitory factor, macrophage-colony
stimulating factor, nerve growth factor, prostaglandin E2,
transforming growth factor-
, and TNF-
), only two cytokines,
TNF-
and IL-1, were found to strongly activate virus transcription
(29). Thus, specific cytokines and/or modulatory factors present in the
placental microenvironment while controlling cellular activities may
also play a regulatory role in protecting the host or, inversely, in
driving HIV-1 expression. More specifically, because HIV-1 may be
present in too low a concentration and/or may be latent, some of these
factors could be, in part, the triggering element necessary for the
expression of HIV-1 in infected trophoblastic cells. This would
translate into productive viral expression and spreading of the virus
to the fetus.
and IL-1
triggered an important increase in viral gene expression. However,
production of progeny virions upon treatment with these
pro-inflammatory cytokines was only seen following co-cultivation with
susceptible indicator cells. These data represent further evidence that
the natural low permissiveness of trophoblasts to productive HIV-1
infection is associated with limitations in the early events of the
virus life cycle. Our results suggest that the presence of cytokines
such as TNF-
and IL-1 in the vicinity of trophoblastic cells in
association with lymphocytic cells would create favorable conditions
leading to vertical transmission of this retrovirus. Considering that
expression of these cytokines is highly regulated according to the
stage of placental development, it can be proposed that windows of
opportunity are transiently created for the induction of viral
expression by extracellular factors in trophoblasts. Collectively, the
data presented may explain in part the mechanism of transmission of
HIV-1 to the fetus during gestation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
R+ vector was constructed by inserting a
frameshift mutation near the env gene and the firefly
luciferase reporter gene into the nef gene (32, 33). Both
molecular constructs were obtained through the AIDS Repository Reagent
Program. The pHCMV-G molecular construct codes for the broad-host-range
vesicular stomatitis virus envelope glycoprotein G (VSV-G) under the
control of the human cytomegalovirus promoter (34). The
pcDNA-1/Amp-based expression vector coding for the HIV-1 Ada-M
(M-tropic) full-length envelope protein was generously provided by
N. R. Landau (The Salk Institute for Biological Studies, La Jolla,
CA). pHXB2-env is a mammalian expression vector coding for
the HIV-1 HXB2 (T-tropic) envelope glycoprotein and was obtained from
the AIDS Repository Reagent Program. pLTR-Luc and pm
BLTR-Luc have
been kindly provided by Dr. K. Calame (Columbia University, New York).
These molecular constructs contain the luciferase reporter gene under
the control of wild-type (GGGACTTTCC) or NF-
B-mutated
(CTCACTTTCC) HIV-1HXB2 LTR (453 to
+80) (35). The molecular construct pNF-
B-Luc contains five (5)
consensus sequences of NF-
B-binding sites placed in front of the
luciferase reporter gene (Stratagene, La Jolla, CA). The reporter gene
vectors pBlue3'LTR-Luc-A to G carry the HIV-1 LTR regions from subtypes
A to G driving the firefly luciferase reporter gene (obtained through
the AIDS Repository Reagent Program) (36).
R+ along
with a vector coding for VSV-G, Ada-M-env, or
HXB2-env to produce the luciferase expressing single cycle
pseudotyped HIV-1 virions. Fully infectious viral entities were
produced by transiently transfecting 293T cells with the infectious
molecular clone pNL4-3 or by co-transfection with pNL4-3 and pHCMV-G.
Sixteen hours after transfection, the cells were washed twice with
phosphate-buffered saline (PBS) and incubated for 24 h in complete
DMEM culture medium. Pseudotypes and fully infectious viruses were
collected at this point by filtering the culture media through a
0.22-µm pore size cellulose acetate membrane (Millipore, Bedford,
MA). Virus stocks were aliquoted and frozen at
85 °C for future
use. All virus preparations underwent only one freeze-thaw cycle before
initiation of infection studies. Virus stocks were normalized for
virion content by using a p24 antibody capture assay developed in our laboratory (39).
(R&D Systems, Minneapolis, MN) and interleukin
(IL)-1
or IL-1
(NCI, National Institutes of Health, Bethesda). After an 8- to 72-h incubation period, luciferase
activity was monitored in cell lysates as described previously (40). For co-culture experiments, 1 × 106 JAR cells were
incubated in 25-cm2 tissue culture dishes at 37 °C for
7 h with fully competent NL4-3 particles (400 ng of p24). Cells
were then washed three times with PBS, trypsinized, and subcultured at
1 × 105 cells per well in 12-well flat-bottom tissue
culture plates in 1.3 ml of complete DMEM culture medium. After an
overnight incubation, 650 µl of culture medium was removed from each
well and replaced with 650 µl of fresh complete DMEM culture medium
supplemented with TNF-
at a final concentration of 10 ng/ml. After a
24-h stimulation period, the infected JAR cells were co-cultured for 24 h with 4 × 105 indicator cells
(i.e. LuSIV or PBMCs). Indicator cells that remained in
suspension were removed from the attached JAR cells, centrifuged for 5 min at 1,200 rpm, and resuspended in the appropriate culture medium.
The rescued LuSIV and PBMCs were seeded in 48-well plates at 3 × 105 cells per well. On each subsequent day, 100 µl of
LuSIV or 150 µl of cell-free culture media from the PBMCs were
transferred to 96-well plates and lysed. The lysed cells and culture
media were frozen. Finally, luciferase activity and p24 level were
assessed in lysed LuSIV and in PBMCs culture media, respectively.
-galactosidase vector (pActin-
-galactosidase) to
normalize for transfection efficiency. Sixteen hours after
transfection, the cells were washed twice with PBS and incubated for
8 h in complete DMEM culture medium. The transfected cells were
then washed three times with PBS, trypsinized, and subcultured at
25 × 103 cells per well in 96-well flat-bottom tissue
culture plates or at 50 × 103 cells per well in
48-well flat-bottom tissue culture plates in 200 µl of complete DMEM
culture medium. After an overnight incubation, 100 µl of medium was
removed from each well and replaced with 100 µl of fresh complete
DMEM culture medium supplemented with TNF-
at a final concentration
of 10 ng/ml. After an 8-h stimulation period, 100 µl of cell-free
culture media was removed, and 25 µl of a 5× luciferase assay lysis
buffer was added. Luciferase activity was measured as described above.
-Galactosidase assays were performed using the Galacto
LightTM chemiluminescent reporter assay for
-galactosidase (Tropix, Bedford, MA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Susceptibility of trophoblastic cell lines to HIV-1 infection with
Ada-M, HXB-2, and VSV-G pseudotypes
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Fig. 1.
HIV-1 enters massively in trophoblastic
cells. JAR cells were exposed to VSV-G pseudotypes (A),
Ada-M pseudotypes (B), or complete NL4-3 viruses
(C) for 5 min to 4 h at 37 °C. The cells were
then washed, trypsinized, and lysed. The process of virus entry was
measured by evaluating the amount of p24 in the cell lysates. Data
shown are expressed as the means ± S.D. of triplicate samples and
are representative of three independent experiments.
View larger version (18K):
[in a new window]
Fig. 2.
Intracellular p24 is primarily localized in
endosomal vesicles in trophoblastic cells. JAR (A) and
PM1 cells (B) were exposed to HIV-1 particles pseudotyped
with Ada-M envelope glycoproteins for the indicated times at 37 °C.
After viral exposure, cells were washed, trypsinized, and resuspended
in a swelling buffer. Cells were next disrupted by Dounce
homogenization, and levels of p24 were evaluated in each cellular
fraction. Data shown are expressed as the means ± S.D. of
triplicate samples and are representative of three independent
experiments.
, IL-1
, and IL-1
--
Based on our
present data, we now know that HIV-1 is indeed present in the cytoplasm
of trophoblastic cells, albeit at a low concentration. We therefore
argue that these viruses can lead to a productive infection. This
notion is controversial because other scientists have failed to detect
the replication of HIV-1 in trophoblastic cells. Two hypotheses can be
made as follows: 1) either the virus is integrated and becomes latent,
and/or 2) the virus is present in such a low concentration in the
cytoplasm that viral expression resulting from these viruses may be too low to be detected. In both instances, external stimulus may be required to detect viral gene expression. Numerous extracellular soluble factors are present in the vicinity of the placenta during gestation, and some of them are known to modulate the expression of
HIV-1 in other cellular environments. We tested the effect of TNF-
,
IL-1
, and IL-1
to trigger the viral regulatory elements in
trophoblastic cells by using recombinant luciferase-expressing HIV-1
pseudotyped with envelope glycoproteins from the Ada-M or HXB2 strain
of HIV-1. In agreement with our previous findings, no increase in
luciferase activity could be detected in untreated JAR cells following
infection with Ada-M or HXB2 pseudotypes. However, a significant
enhancement of HIV-1 LTR activity was observed upon
exposure of such virally infected JAR cells to TNF-
and IL-1
(Fig. 3). For example, incubation of
Ada-M-infected JAR cells with increasing doses of TNF-
and IL-1
led to a maximal 87.3- and 26.3-fold increase in LTR activity,
respectively. A similar treatment of JAR cells once infected with HXB2
pseudotypes resulted also in induction of HIV-1
LTR-dependent reporter gene activity but to a smaller
extent. Similar observations were made when JAR cells were treated
instead with IL-1
(data not shown). Further studies revealed that
the positive effect of TNF-
, IL-1
, and IL-1
on virus
transcriptional activity was dose-dependent (Fig.
4). To demonstrate that the noticed
effect was not cell type-specific, we tested the effect of TNF-
,
IL-1
, and IL-1
on two other trophoblastic cell lines,
i.e. BeWo and JEG-3. Following virus infection, HIV-1
LTR-driven luciferase expression was enhanced in a
dose-dependent manner by the three tested cytokines in BeWo cells (Fig. 5). The highest levels of
reporter gene activity were seen following treatment of BeWo cells with
TNF-
(i.e. 8.2-fold increase with the highest dose of
TNF-
). Exposure of BeWo cells to IL-1
and IL-1
produced a
4-fold increase in HIV-1 LTR activity. Similar data were obtained when
using JEG-3 cells (data not shown).
View larger version (18K):
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Fig. 3.
HIV-1 transcriptional activity is increased
upon treatment of trophoblasts with TNF- and
IL-1
. JAR cells were infected with HIV-1 reporter
viruses bearing M-tropic (A and B) or T-tropic
(C and D) envelope glycoproteins and were either
left untreated or treated with increasing doses of TNF-
(A and C) or IL-1
(B and
D). Cells were lysed at 24 h post-stimulation, and
HIV-1-encoded luciferase activity was monitored in each cell lysate.
Values from the luminometer are expressed as relative light units
(RLU). Data shown are expressed as the means ± S.D. of
quadruplicate samples and are representative of three independent
experiments. Fold increase over untreated cells is indicated at the top
of each data point.
View larger version (9K):
[in a new window]
Fig. 4.
Cytokine-mediated increase in HIV-1 LTR
activity is dose-dependent in trophoblasts. JAR cells
were first infected with HIV-1 particles bearing VSV-G envelope
glycoproteins and were either left untreated or treated with increasing
doses of TNF- (A), IL-1
(B), or IL-1
(C). Cells were lysed either at 8 (C) or 24 h (A and B) post-stimulation, and HIV-1-encoding
luciferase activity was assessed in each cell lysate. Values from the
luminometer are expressed as relative light units × 103 (kRLU). Data shown are expressed as the
means ± S.D. of quadruplicate samples and are representative of
three independent experiments.
View larger version (11K):
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Fig. 5.
Cytokine-mediated induction of HIV-1
transcription is also seen in BeWo trophoblastic cells. BeWo cells
were inoculated with VSV-G pseudotypes and were either left untreated
or treated with TNF- (A), IL-1
(B), or
IL-
(C). Cells were lysed at 24 h post-stimulation,
and HIV-1-encoded luciferase activity was monitored in each cell
lysate. Values from the luminometer are expressed as relative light
units × 103 (kRLU). Data shown are
expressed as the means ± S.D. of quadruplicate samples and are
representative of three independent experiments. Fold increase over
untreated cells is indicated at the top of each bar.
and Co-culture with
Indicator Cells--
In order to investigate whether the
cytokine-mediated up-regulation of HIV-1 transcriptional activity could
be seen in the context of the complete viral genome, JAR cells were
inoculated with fully infectious HIV-1NL4-3 viruses before
exposure to TNF-
. Virus production was found to be near the
detection limit of our p24 enzymatic assay (i.e. less than
50 pg/ml) despite exposure to TNF-
(data not shown). We next
attempted to rescue the seemingly low amount of produced viruses by
co-cultivation with the CXCR4-expressing LuSIV cell line. Previous
experiments performed in our laboratory suggest that such indicator
cells are susceptible to infection with very low levels of HIV-1,
i.e. less than 1 pg of
p24.2 No noticeable increase
in luciferase activity was obtained in LuSIV co-cultured
with virus-infected JAR cells that were left unstimulated (Fig.
6A). However, the
co-cultivation of LuSIV cells with TNF-
-treated JAR cells that were
initially infected with NL4-3 resulted in a dramatic augmentation of
luciferase activity. To more closely parallel the physiological
conditions, similar studies were carried out using PBMCs as indicator
cells. When PBMCs were co-cultured with unstimulated virus-infected JAR
cells, virus production was again below the detection limit of our p24 assay (Fig. 6B). Interestingly, virus production was rescued
when PBMCs were instead co-cultured with NL4-3-infected JAR cells that were also treated with TNF-
. Again, these data fully support the
notion that human trophoblast cells produced only low amounts of mature
virus progeny, levels that can be significantly augmented upon
treatment with a pro-inflammatory cytokine such as TNF-
.
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Fig. 6.
Production of fully infectious HIV-1
particles is seen when virus-infected trophoblasts are treated with
TNF- and co-cultured with indicator
cells. JAR cells were inoculated with fully infectious NL4-3
virions and were either left untreated or treated with TNF-
at 10 ng/ml. The infected JAR cells were next co-cultured for 24 h with
indicator LuSIV cells (A) or primary human PBMCs
(B). Luciferase activity in the indicator LuSIV cell line
was measured on days 3, 5, 7, 9, and 12 post-coculture (A),
whereas levels of p24 were evaluated in cell-free culture media from
PBMCs on days 1, 3, 5, 7, 9, and 12 post-coculture
(B). Luciferase activity is depicted as relative light
units × 103 (kRLU). Data shown are
expressed as the means ± S.D. of quadruplicate samples and are
representative of three independent experiments.
. This technical strategy
permits the bypassing of the natural inefficient route of HIV-1 entry
in trophoblasts and ultimately to the release of complete HIV-1
particles. In JAR cells infected with complete NL4-3 virions
pseudotyped with VSV-G envelope glycoproteins, the release of a
significant amount of p24 was detected throughout the course of
infection, and virus production was again markedly augmented by a
treatment with TNF-
(Fig. 7). Thus no
intracellular restriction is present in trophoblastic cells, and
together these studies suggest that the route of HIV-1 entry is the
major limiting step of HIV-1 infection in trophoblasts.
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Fig. 7.
Limited HIV-1 infection in trophoblast cells
is not related to intracellular restrictions other than the route of
virus entry. JAR cells were inoculated with complete NL4-3 virions
pseudotyped with VSV-G envelope glycoproteins and were left untreated
or treated with TNF- at 10 ng/ml. Cell-free culture media were
collected on days 1, 2, 3, 5, and 7 days post-stimulation, and levels
of p24 were evaluated. Levels of p24 were monitored in cell-free
culture media at the indicated times. Data shown are expressed as the
means ± S.D. of quadruplicate samples and are representative of
three independent experiments.
and IL-1
in Trophoblasts--
To assess whether changes in
the LTR architecture due to the recognized genetic heterogeneity of
HIV-1 can influence the cytokine-mediated modulatory effect seen in
trophoblast cell lines, transfection experiments were conducted with
molecular constructs made of various LTR subtypes. JAR cells were
transiently transfected with luciferase-encoding vectors carrying the
LTR region from clades A to G before treatment with TNF-
and
IL-1
. As shown in Fig. 8, virus
transcription was triggered by both TNF-
and IL-1
for all the
HIV-1 LTR clades tested. This indicates that the up-regulating effect
of these cytokines on HIV-1 transcription is not restricted to a
specific HIV-1 LTR subtype.
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Fig. 8.
Different HIV-1 LTR subtypes are
transactivated by TNF- and
IL-1
in trophoblasts. JAR cells were
co-transfected with an expression vector coding for the luciferase
reporter gene under the control of an HIV-1 LTR subtype and an
actin-driven
-galactosidase vector. Cells were next either left
untreated or treated with TNF-
or IL-1
and were lysed at 8 h
post-stimulation. HIV-1-encoded luciferase activity and
actin-dependent
-galactosidase values were monitored in
each cell lysate. Standardization of the luciferase counts was achieved
by dividing each luciferase activity mean value by the measured
-galactosidase activity mean value. Data shown are from
quadruplicate samples and are representative of three independent
experiments. Fold increase over untreated cells is indicated at the
top of each bar.
B--
Finally, we investigated
the molecular events leading to TNF-
-induced transactivation of
HIV-1 by transiently transfecting the JAR cell line with various
expression vectors before exposure to TNF-
. As depicted in Fig.
9, TNF-
triggered a 4.4-fold increase in luciferase activity in JAR cells transfected with the luciferase reporter gene placed under the control of wild-type
HIV-1HXB2 LTR when compared with untreated JAR cells.
However, this induction was lost when JAR cells were transfected with a
vector that bears mutations at the two NF-
B-binding sites within the
HIV-1HXB2 LTR domain. The involvement of NF-
B in this
process was confirmed by the observation that a 7.4-fold increase in
luciferase activity was obtained upon the addition of TNF-
to JAR
cells that were transfected with a
B-driven reporter gene
vector.
View larger version (14K):
[in a new window]
Fig. 9.
Cytokine-dependent
transactivation of HIV-1 LTR in trophoblasts is mediated via
NF- B. JAR cells were transiently
transfected with pLTR-Luc, pm
BLTR-Luc, or pNF
B-Luc and were
either left untreated or treated with TNF-
. Cells were lysed at
8 h post-stimulation, and luciferase activity was monitored in
each cell lysate. Values from the luminometer are expressed as relative
light units (RLU). Data shown are expressed as the
means ± S.D. of quadruplicate samples and are representative of
three independent experiments. Fold increase over untreated cells is
indicated at the top of each bar.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, IL-1
, and IL-1
were found
to act as potent inducers of HIV-1 LTR-dependent activity in trophoblasts when these cells were infected with VSV-G pseudotypes. Interestingly, the same cytokines also induced viral transcription in
trophoblastic cells exposed to replication-defective reporter viruses
bearing HIV-1 Ada-M or HXB2 envelope glycoproteins. These findings are
supported by a recent study (44) showing that HIV-1 LTR activity is
increased by TNF-
and IL-1
in primary isolated human trophoblast
cells that were transiently transfected with an HIV-1-based reporter
vector. Data from this study were entirely based on transient
transfection of HIV-1 LTR-based reporter constructs in trophoblast
cells. In contrast, our observations are founded on integrated
proviruses, i.e. following infection of trophoblasts with
either complete HIV-1 particles or HIV-1-based pseudotyped viruses.
This is important to note because recent findings indicate that
integrated proviral DNA behaves quite differently from transfected plasmids. Indeed, expression of integrated human retroviruses such as
HIV-1 and HTLV-1 has been shown to require cellular factors different
from those necessary for expression of transiently transfected retroviral based plasmids (45-48). More specifically, it has been demonstrated that transcription from integrated versus
transiently introduced retroviral LTRs does not proceed via identical
signal transduction pathways. This suggests that the signaling
requirements necessary to achieve expression of an integrated proviral
DNA can differ from a transfected viral plasmid. Because the process of
integration into the host chromosome is an obligatory step in HIV-1
life cycle, the series of investigations that we performed with
integrated HIV-1 provide physiological significance to our work.
and/or IL-1. These newly
released virions, as we observed when HIV-1-infected trophoblasts were
treated with TNF-
and co-cultured with PBMCs, could next
productively infect underlying susceptible fetal cells including
Hofbauer cells (49).
and IL-1
during vertical transmission of HIV-1 has been indirectly put forward
in previous studies. First, trophoblastic cells from HIV-1-infected
placentas were found to express higher levels of TNF-
, IL-1
, and
IL-6, and such levels also correlated with the amounts of HIV-1 Gag
transcripts found in trophoblastic cells (49, 50). Second, pretreatment
of trophoblastic cells with TNF-
and IL-1
increased lymphocytic
cell adhesion to trophoblastic cells (51). Third, cell contact between
macrophages and trophoblasts resulted in up-regulation of HIV-1
expression that was mediated by the release of TNF-
and IL-6 by
macrophages (52). Based on these published data together with the
present findings, it can be proposed that TNF-
and IL-1 are produced
by HIV-1-infected trophoblastic cells and/or macrophages upon cell
contact with trophoblasts. As a consequence, these cytokines would in
turn up-regulate HIV-1 LTR activity and virus production. This may represent a potential mechanism of HIV-1 replication in trophoblastic cells leading ultimately to vertical transmission.
was able to activate HIV-1 LTR-driven transcriptional activity
in trophoblast cells at a concentration as low as 1 ng/ml. The
physiological significance of such findings is provided by the
demonstration that TNF-
was reported to range from 1.1 to 2.8 ng/ml
in placental supernatants and from 3.9 to 8.5 ng/ml in decidual
supernatants (53). IL-1
and IL-1
, on the other hand, triggered
significant activation of the regulatory elements of HIV-1 at a
concentration up to a 100-fold less than TNF-
. Considering that IL-1
was previously reported to be at a concentration of 0.19 ng/ml at term
and 0.68 ng/ml at the onset of labor (54), it can be proposed that IL-1
is also an extremely potent activator of the LTR of HIV-1 in
trophoblastic cells. Moreover, the strong effect mediated by TNF-
and IL-1 on LTR activity was neither cell line- nor clade-specific,
therefore providing credence to the observed phenomenon. At the
molecular level, the cytokine-mediated augmentation of HIV-1
transcriptional activity appears to be mediated via the ubiquitous
mammalian transcriptional factor NF-
B. The TNF-
-dependent signaling pathway has been extensively
studied in lymphocytic and monocytic cells, and NF-
B is thought to
play a key role in triggering HIV-1 expression in these cells, which are considered as major cellular reservoir of this retrovirus (28).
during gestation are thought to include the
following: control of cell migration and placental growth, cell death,
immune privilege, hormone production (progesterone, estradiol, and
human chorionic gonadotropin), and parturition (55-57). It is
interesting to note that both TNF-
and IL-1 exert their effect
primarily at the onset of pregnancy and again during labor (25, 53,
58-65). Interestingly, it coincides with the timing of a higher risk
of HIV-1 vertical transmission (7, 11). Moreover, concomitant viral and
bacterial infections are among the risk factors associated with
vertical transmission of HIV-1 (66). Given that certain microbial
infections lead to a transient expression of IL-1 and TNF-
during
pregnancy (67), it is tempting to speculate that the presence of
pro-inflammatory cytokines creates conditions leading to higher HIV-1
expression and thus an increased probability of its vertical transmission.
and IL-1. The
expression profile of these cytokines suggests that productive HIV-1
infection of trophoblasts may be favored at two different time points
during the course of pregnancy, i.e. first at the onset of
pregnancy, where there are highly proliferative and invasive
trophoblastic cells, and later at term. Because there is a limited time
frame of TNF-
and IL-1 expression and a bias toward Th2-type
cytokine expression during pregnancy, this may help to explain in part why HIV-1 transmission may be limited during pregnancy.
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ACKNOWLEDGEMENT |
---|
We thank Dr. M. Duffer for technical assistance in flow cytometry studies.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Canadian Institutes of Health Research HIV/AIDS Research Program Grant HOP-15575 (to M. J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Performed this work in partial fulfillment for the Ph.D. Degree in
the Program of Microbiology-Immunology, Faculty of Medicine, Laval University.
§ Recipient of Canadian Institutes of Health Research Doctoral Research Awards from the HIV/AIDS Research Program.
¶ Holds a Tier 1 Canada Research Chair in Human Immuno-Retrovirology. To whom correspondence should be addressed: Laboratoire d'Immuno-Rétrovirologie Humaine, Centre de Recherche en Infectiologie, RC709, Hôpital CHUL, Centre Hospitalier Universitaire de Québec, 2705 Blvd. Laurier, Ste-Foy, Québec G1V 4G2, Canada. Tel.: 418-654-2705; Fax: 418-654-2212; E-mail: michel.j.tremblay@crchul.ulaval.ca.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M210470200
2 G. Vidricaire, M. R. Tardif, and M. J. Tremblay, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HIV-1, human
immunodeficiency virus type-1;
IL, interleukin;
TNF-, tumor necrosis
factor-
;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal
bovine serum;
PBS, phosphate-buffered saline;
PBMCs, peripheral blood
mononuclear cells;
VSV, vesicular stomatitis virus;
LTR, long terminal
repeat;
RLU, relative light units.
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