The low viral production in trophoblastic cells

is due to a high endocytic internalization of the human immunodefiency virus type 1

and can be overcome by the pro-inflammatory cytokines TNF-α and IL-1

Gaël Vidricaire, Mélanie R. Tardif and Michel J. Tremblay*

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, Canada, G1V 4G2

*Corresponding author. Mailing address:

Laboratoire d'Immuno-Rétrovirologie Humaine

Centre de Recherche en Infectiologie, RC709

Hôpital CHUL, Centre Hospitalier Universitaire de Québec

2705 boul. Laurier

Ste-Foy,Québec, Canada, G1V 4G2

Phone: (418) 654-2705; Fax: (418) 654-2212

E-mail: michel.j.tremblay@crchul.ulaval.ca

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ABSTRACT

Maternal-infant transmission of human immunodeficiency virus type-1 (HIV-1) is the primary

cause of this retrovirus infection in neonates. Although the mechanism(s) through which HIV-1

crosses the placenta to reach the fetus remains unclear, trophoblasts have been proposed to play a

critical role in modulating virus spread to the fetus. Thus, 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. Interestingly,

HIV-1 transcription and virus production of infectious particles were both augmented upon

treatment of trophoblasts with TNF-α and IL-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 due to a restriction in the mode of entry and such

a limitation can be overcome with physiologic doses of TNF-α and IL-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 environmental milieu may

thus contribute to the propagation of HIV-1 across the placenta.

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INTRODUCTION

Mother-to-child transmission of the human immunodeficiency virus type 1 (HIV-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 (1)). In the absence of

antiretroviral treatment, the reported risk of vertical transmission lies within the range of 10 to

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 infants’ skin and

mucous membrane to maternal blood and vaginal secretion) (4-6). Mathematical modeling has

allowed estimating that 30% of infections occur in utero less than two 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 foetuses and in second trimester fetal tissues (8-11).

In order for the virus to reach the fetal circulation and infect the foetus in utero, HIV-1 must

cross the placental barrier which is made of a double layer of polarized epithelial-type cells, the

cytotrophoblasts and syncytiophoblasts. 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 foetus. It has been shown that the placenta

may allow transcytosis of the virus from the maternal to the foetal circulation (12). Alternatively

and/or concomitantly, the virus may infect the placenta to reach the fetal circulation and

ultimately the foetus. Indeed, HIV-1 has been detected on both the maternal and foetal 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

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evolutionary relationships of HIV-1’s sequences 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 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 HIV-1’s phenotype in

conjunction with particular cellular environments. This may include limitations in virus entry,

intra-cellular restriction, inappropriate cellular environment or a combination of the three

(reviewed in (20)). The question of viral entry is crucial since the expression of HIV-1’s cellular

receptor CD4 is very low whereas the expression of HIV-1’s co-receptors, CXCR4 and CCR5,

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, GM-CSF, M-CSF, LIF, TGF-β, IFN-γ and TNF-α), hormones, growth factors (e.g.

EGF, VEGF, progesterone and oestrogen) and prostaglandins (e.g. PGE2) 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 (25-27)). On the other hand,

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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 (20, 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

trophoblats. Among all the soluble agents tested (i.e. EGF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-3,

IL-4, IL-6, IL-7, IL-8, IL-10 IL-12, LIF, M-CSF, NGF, PGE2, TGF-β and TNF-α), only two

cytokines, TNF-α 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, since HIV-1 may be present in too low

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 foetus.

The central objective of the present work was to provide further insight on the susceptibility of

human trophoblast 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 since we found massive HIV-1 entry in trophoblastic cells.

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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-α 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-α 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 HIV-1’s transmission to the foetus

during gestation.

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MATERIALS AND METHODS

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, Ca). The JAR, JEG-3 and

293T cells lines were cultured in DMEM medium (Gibco-BRL, Burlington, Canada) whereas the

BeWo cell line was maintained in F12 Nutrient Mixture (Gibco-BRL, Burlington, Canada). Both

media were supplemented with 10% fetal bovine serum (FBS) (Invitrogen Life Technologies,

Burlington, Canada), glutamine (2 mM), penicillin G (100 U/ml) and streptomycin (100 mg/ml).

BeWo, JAR and JEG-3 cells were routinely subcultured at a seeding density of 3 x 106 cells and

293T cells at 1 x 106 cells in 75 cm2 tissue culture flasks. PM1 cells were obtained through the

AIDS Research and Reference Reagent Program (Division of AIDS, National Institute of Allergy

and Infectious Diseases, Rockville, MD). These cells were cultured in RPMI 1640 medium

supplemented with 10% FBS. The reporter LuSIV cell line, kindly provided by J. E. Clements

(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 x 106 cells/ml in selection medium

made of RPMI 1640 (Gibco-BRL, Burlington, Canada) with 10% FBS, 15 mM NAOH, 25 mM

HEPES, and supplemented with glutamine (2 mM), penicillin G (100 U/ml), streptomycin (100

mg/ml) and 300 µg/ml hygromycin B (Boehringer Ingelheim, Indianapolis, IN). Human

peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by centrifugation

through a Ficoll-Hypaque 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

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(2 mM), penicillin G (100 U/mL), streptomycin (100 mg/ml), 3 µg/ml of PHA-P (Sigma, St-

Louis, Mo) and 50 U/ml of human recombinant IL-2 (kind gift of M. Gately, Hoffman-Laroche

Inc.) (31).

Molecular constructs. pNL4-3 is a full-length infectious molecular clone of HIV-1 while the

NL4-3-Luc E-R+ vector was constructed by inserting a frame shift 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 pmκBLTR-Luc

have been kindly provided by Dr. K. Calame (Columbia University, New York, NY). These

molecular constructs contain the luciferase reporter gene under the control of wild-type

(GGGACTTTCC) or NF-κB-mutated (CTCACTTTCC) HIV-1HXB2 LTR (453 to +80) (35). The

molecular construct pNF-κB-Luc contains five (5) consensus sequences of NF-κB 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 subtype A, B, C, D, E, F,

and G driving the firefly luciferase reporter gene (obtained through the AIDS Repository Reagent

Program) (36).

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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 to 80% confluence the day of transfection. Using the Clontech

Transfection Kit (BD Biosciences Clonetech, Palo Alto, CA), cells were co-transfected with

NL4-3-Luc E-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).

Virus entry and cell fractionation assays. Approximately 1 x 106 of JAR and PM1 cells were

incubated with Aad-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 previously described

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(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 to 6 x 106 BeWo, JAR, or JEG-3 cells were incubated at 37ºC under a 5% CO2

atmosphere for 7 h with luciferase-encoding HIV-1 particles pseudotyped with VSV-G (6 to 145

ng of p24), Ada-M, or HXB2 envelope glycoproteins (250 to 400 ng of p24). Cells were then

washed three times with PBS, trypsinized and subcultured at 25 x 103 cells per well in 96-well

flat-bottom tissue culture plates or at 50 x 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)-α (R&D Systems, Minneapolis, MN),

interleukin (IL)-1α or IL-1β (National Cancer Institute, Rockville, MD). After an 8 to 72 h

incubation period, luciferase activity was monitored in cell lysates as previously described (40).

For co-culture experiments, 1 x 106 JAR cells were incubated in 25 cm2 tissue culture dishes at

37ºC for 7 h with fully competent NL4-3 particles (400 ng of p24). Cells were then washed three

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times with PBS, trypsinized and subcultured at 1 x 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-α 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 x 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 x 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.

Transient transfection of JAR cells. JAR cells were transiently transfected by calcium

phosphate precipitation using the Clontech Transfection Kit. Briefly, 0.5 to 1.5 x 106 JAR cells

were plated 16 h before transfection in 25 cm2 tissue culture dishes. For each transfection, 5 µg of

plasmid DNA were used. For the HIV-1 Clades A to G transfection experiment, JAR cells were

co-transfected with 1 µg of an actin promoter-driven β-galactosidase vector (pActin-β-

galactosidase) to normalize for transfection efficiency. Sixteen h 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 x 103

cells per well in 96-well flat-bottom tissue culture plates or at 50 x 103 cells per well in 48-well

flat-bottom tissue culture plates in 200 µl of complete DMEM culture medium. After an

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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-α at a final concentration of 10

ng/ml. After a 8 h stimulation period, 100 µl of cell-free culture media were removed and 25 µl

of a 5X luciferase assay lysis buffer were added. Luciferase activity was measured as described

above. β-galactosidase assays were performed using the Galacto Light™ chemiluminescent

reporter assay for β-galactosidase (Tropix, Bedford, MA).

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RESULTS

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 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 (30). 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 1). 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 with 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 since 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 370C.

Subsequently, the cells were trypsinized, washed and lysed before monitoring the amount of

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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 period of 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.

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HIV-1 LTR-driven reporter gene activity is increased in trophoblastic cells by TNF-α, IL-1α

and IL-1β. 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 since other scientists have

failed to detect HIV-1’s replication in trophoblastic cells. Two hypotheses can be made: 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 extra-cellular soluble factors are present in the vicinity of the placenta

during gestation and some of them are known to modulate HIV-1’s expression in other cellular

environment. We tested the effect of TNF-α, IL-1α and IL-1β to trigger the viral regulatory

elements in trophoblastic cells by using recombinant luciferase-expressing HIV-1 pseudotyped

with envelope glycoproteins from 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-α and

IL-1β (Fig. 3). For example, incubation of Ada-M-infected JAR cells with increasing doses of

TNF-α and IL-1β 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-1α (data not shown). Further studies revealed

that the positive effect of TNF-α, IL-1α and IL-1β on virus transcriptional activity was dose

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dependent (Fig. 4). To demonstrate that the noticed effect was not cell type-specific, we tested the

effect of TNF-α, IL-1α and IL-1β 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-α (i.e. 8.2-fold

increase with the highest dose of TNF-α. Exposure of BeWo cells to IL-1α and IL-1β produced a

4-fold increase in HIV-1 LTR activity. Similar data were obtained when using JEG-3 cells (data

not shown).

Production of fully infectious HIV-1 particles by trophoblastic cells is only seen following

treatment with TNF-α 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-α. 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-α (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 (unpublished observations). 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-α-treated JAR cells that were initially

infected with NL4-3 resulted in a dramatic augmentation of luciferase activity. To more closely

parallel physiological conditions, similar studies were carried out using PBMCs as indicator cells.

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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-α. 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-α.

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-α. This technical strategy permits to

bypass 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-α (Fig. 7). Thus no intracellular restriction is present in trophoblatic cells and

together these studies suggest that the route of HIV-1 entry is the major limiting step of HIV-1

infection in trophoblasts.

Different HIV-1 LTR subtypes are transactivated by TNF-α and IL-1β in trophoblasts. To

assess whether changes in the LTR architecture due to the recognized genetic heterogeneity of

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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 LTR

region from clades A through G before treatment with TNF-α and IL-1β. As shown in Fig. 8,

virus transcription was triggered by both TNF-α and IL-1β 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.

Cytokine-dependent up-regulation of HIV-1 LTR activity in trophoblasts is mediated via NF-

κB. Finally, we investigated the molecular events leading to TNF-α-induced transactivation of

HIV-1 by transiently transfecting the JAR cell line with various expression vectors before

exposure to TNF-α. As depicted in Fig. 9, TNF-α 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 to untreated JAR cells. However, this induction was

lost when JAR cells were transfected with a vector that bears mutations at the two NF-κB-binding

sites within the HIV-1HXB2 LTR domain. The involvement of NF-κB in this process was

confirmed by the observation that a 7.4-fold increase in luciferase activity was obtained upon the

addition of TNF-α to JAR cells that were transfected with a κB-driven reporter gene vector.

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DISCUSSION

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 environmental milieu 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

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consequence 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 since

viruses will ultimately be degraded in lysosomes (43). In trophoblastic cells, phagocytosis is

thought to play an important role in the extensive tissue remodelling that occurs during

trophoblastic invasion of the deciduas and in the control of exchanges between the maternal and

foetal 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-α, IL-1α and IL-1β 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 showing that HIV-1 LTR activity is

increased by TNF-α and IL-1β in primary isolated human trophoblast cells that were transiently

transfected with an HIV-1-based reporter vector (44). 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

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note since 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 factor 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. Since 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.

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 since 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 in spite of 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-α and/or IL-1.

These newly released virions, as we observed when HIV-1-infected trophoblasts were treated

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with TNF-α and co-cultured with PBMCs, could next productively infect underlying susceptible

fetal cells including Hofbauer cells (49).

In line with our results, the relative importance of TNF-α and IL-1 during vertical

transmission of HIV-1 has been indirectly put forward in previous works. First, trophoblastic

cells from HIV-1-infected placentas were found to express higher levels of TNF-α, IL-1β and IL-

6 and such levels also correlated with the amounts of HIV-1 Gag transcripts found in

trophoblastic cells (49, 50). Second, pre-treatment of trophoblastic cells with TNF-α and IL-1β

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-α and IL-6 by macrophages (52). Based on these published data together

with the present findings, it can be proposed that TNF-α 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.

TNF-α 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-α 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-1α and IL-1β, 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-α. Considering that IL-1 was previously reported to be at a

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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 HIV-1’s LTR in trophoblastic cells. Moreover,

the strong effect mediated by TNF-α 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-κB. The TNF-α-dependent signalling pathway

has been extensively studied in lymphocytic and monocytic cells and NF-κB is thought to play a

key role in triggering HIV-1 expression in these cells, which are considered as major cellular

reservoirs of this retrovirus (28).

The roles played by TNF-α during gestation are thought to include: control of cell migration

and placental growth, cell death, immune privilege, hormone production (progesterone,

oestradiol, hCG) and parturition (55-57). It is interesting to note that both TNF-α and IL-1 exert

their effect primarily at the onset of pregnancy and again during labour (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-α 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.

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-

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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-α and IL-1. The expression profile of these cytokines

suggest that productive HIV-1 infection of trophoblasts may be favoured 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. Since there is a limited time frame

of TNF-α and IL-1 expression and a bias towards Th2-type cytokine expression during

pregnancy, this may help to explain in part why HIV-1 transmission may be limited during

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ACKNOWLEDGEMENTS

We thank Dr. M. Dufour for technical assistance in flow cytometry studies. This study was

supported by a grant to M.J.T. from the Canadian Institutes of Health Research (CIHR)

HIV/AIDS Research Program (Grants #HOP-15575). This work was performed by G.V. in

partial fulfillment of her Ph.D. Degree in the Program of Microbiology-Immunology, Faculty of

Medicine, Laval University. G.V. and M.R.T. are recipients of CIHR Doctoral Research Awards

from the HIV/AIDS Research Program and M.J.T. holds a Tier 1 Canada Research Chair in

Human Immuno-Retrovirology.

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FIGURE LEGENDS

Figure 1. HIV-1 enters massively in trophoblastic cells. JAR cells were exposed to VSV-G

pseudotypes (panel A), Ada-M pseudotypes (panel B), or complete NL4-3 viruses for 5 min to 4

h at 370C. The cells were then washed, trypsinised 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 +/- standard deviations of triplicate samples and are representative of three independent

experiments.

Figure 2. Intracellular p24 is primarily localized in endosomal vesicles in trophoblastic cells.

JAR (panel A) and PM1 cells (panel B) were exposed to HIV-1 particles pseudotyped with Ada-

M envelope glycoproteins for the indicated time periods at 37°C. After viral exposure, cells were

washed, trypsinised 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 +/- standard deviations of triplicate samples and are representative of

three independent experiments.

Figure 3. HIV-1 transcriptional activity is increased upon treatment of trophoblasts with TNF-α

and IL-1β. JAR cells were infected with HIV-1 reporter viruses bearing M-tropic (panels A and

B) or T-tropic (panels C and D) envelope glycoproteins and were either left untreated or treated

with increasing doses of TNF-α (panels A and C) or IL-1β (panels C 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

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expressed as the means +/- standard deviations of quadruplicate samples and are representative of

three independent experiments. Fold increase over untreated cells is indicated at the top of each

data point.

Figure 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-α (panel A), IL-1α (panel B),

or IL-1β (panel C). Cells were lysed either at 8 h (panel C) or 24 h (panels 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 x 103 (kRLU). Data shown are expressed

as the means +/- standard deviations of quadruplicate samples and are representative of three

independent experiments.

Figure 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-α (panel A), IL-1α (panel B), or IL-β (panel 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 x 103 (kRLU). Data shown are expressed

as the means +/- standard deviations of quadruplicate samples and are representative of three

independent experiments. Fold increase over untreated cells is indicated at the top of each bar.

Figure 6. Production of fully infectious HIV-1 particles is seen when virus-infected trophoblasts

are treated with TNF-α and co-cultured with indicator cells. JAR cells were inoculated with fully

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infectious NL4-3 virions and were either left untreated or treated with TNF-α at 10 ng/ml. The

infected JAR cells were next co-cultured for 24 h with indicator LuSIV cells (panel A) or primary

human PBMCs (panel B). Luciferase activity in the indicator LuSIV cell line was measured on

day 3, 5, 7, 9 and 12 post-coculture (panel A), while levels of p24 were evaluated in cell-free

culture media from PBMCs on day 1, 3, 5, 7, 9 and 12 post-coculture (panel B). Luciferase

activity is depicted as Relative Light Units x 103 (kRLU). Data shown are expressed as the means

+/- standard deviations of quadruplicate samples and are representative of three independent

experiments.

Figure 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-α

at 10 ng/ml. Cell-free culture media were collected on day 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 time periods. Data shown are expressed as the means +/- standard deviations of

quadruplicate samples and are representative of three independent experiments.

Figure 8. Different HIV-1 LTR subtypes are transactivated by TNF-α and IL-1β 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 β-galactosidase vector. Cells

were next either left untreated or treated with TNF-α or IL-1β and were lysed at 8 h post-

stimulation. HIV-1-encoded luciferase activity and actin-dependent β-galactosidase value were

monitored in each cell lysate. Standardization of the luciferase counts was achieved by dividing

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34

each luciferase activity mean value by the measured β-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.

Figure 9. Cytokine-dependent transactivation of HIV-1 LTR in trophoblasts is mediated via NF-

κB. JAR cells were transiently transfected with pLTR-Luc, pmκBLTR-Luc, or pNF-κB-Luc and

were either left untreated or treated with TNF-α. 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 +/- standard deviations of

quadruplicate samples and are representative of three independent experiments. Fold increase

over untreated cells is indicated at the top of each bar.

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0

10

20

30

40

50

60

70

80

90

100

5 min 15 min 30 min 1 h 2 h 4 h

Vira

l Ent

ry (

ng/m

l of p

24)

Ada-M

A

B

C

0

5

10

15

20

25

30

35

40

5 min 15 min 30 min 1 h 2 h 4 h

NL4-3

Vira

l Ent

ry (

ng/m

l of p

24)

0

10

20

30

40

50

60

70

80

90

100

5 min 15 min 30 min 1 h 2 h 4 h

VSV-G

Vira

l Ent

ry (

ng/m

l of p

24)

Figure 1

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0

400

800

1200

1600

2000

5 min 30 min 60 min 120 min

JAR

0

400

800

1200

1600

2000

5 min 30 min 60 min 120 min

PM1

A

B

100%97%

82%

70%

63%

65%

p24

(pg/

ml)

p24

(pg/

ml)

Cytosol Vesicles

35%37%30%

18%

Ada-M

Ada-M

Figure 2

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C

0.0

2.0

4.0

6.0

8.0

10.0

0 0.01 0.1 1 10 100

Luciferase Activity (RLU)Lu

cife

rase

Act

ivity

(RLU

) HXB-2 with TNF-α

0.6X 0.6X

4.3X4.2X

4.7X

TNF-α (ng/ml)

0.0

20.0

40.0

60.0

80.0

100.0

120.0

0 0.01 0.1 1 10 100

.0Luciferase Activity (RLU)

Luci

fera

se A

ctiv

ity (R

LU)

A

Ada-M with TNF-α

1.4X 1.1X

18.6X

87.3X

48.5X

TNF-α (ng/ml)

0.0

4.0

8.0

12.0

16.0

20.0

0 0.002 0.02 0.2 2 20

Luciferase Activity (RLU)

Luci

fera

se A

ctiv

ity (R

LU)

B

Ada-M with IL-1β

1.2X5.4X

26.3X22.6X

16.2X

IL-1β (ng/ml)

Luciferase Activity (RLU)

Luci

fera

se A

ctiv

ity (R

LU) HXB-2 with IL-1b

1.5X 1.6X

14.5X

19X

1.5X

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 0.002 0.02 0.2 2 20

D

IL-1β (ng/ml)

HXB-2 with IL-1β

Figure 3

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0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 0.01 0.1 1 10 100

Dose (ng/ml)

TNF-α

Luci

fera

se A

ctiv

ity (k

RLU

)

A

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0 0.02 0.2 2 20 200

Dose (ng/ml)

IL-1β

Luci

fera

se A

ctiv

ity (k

RLU

)

C

0.0

10.0

20.0

30.0

40.0

50.0

0 0.01 0.1 1 10 100

Dose (ng/ml)

IL-1α

Luci

fera

se A

ctiv

ity (k

RLU

)

B

Figure 4

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0.0

0.4

0.8

1.2

1.6

2.0

TNF-α (ng/ml)

0

0.1

1

10Lu

cife

rase

Act

ivity

(kR

LU)

ABeWo

3.5X

5.4X

8.2X

0.0

0.2

0.4

0.6

0.8

1.0

IL-1β (ng/ml)

0

0.02

0.2

2

Luci

fera

se A

ctiv

ity (

kRLU

)

C

BeWo

1.8X

4.2X 4.0X

0.0

0.2

0.4

0.6

0.8

1.0

1.2

IL-1α (ng/ml)

0

0.01

0.1

1

Luci

fera

se A

ctiv

ity (k

RLU

)

BBeWo

2.3X

3.9X

4.4X

Figure 5

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Vira

l Pro

duct

ion

(ng

of p

24)

Luci

fera

se A

ctiv

ity (k

RLU

)

A

B

0.0

3.0

6.0

9.0

12.0

15.0

3 5 7 9 12

Time Course (day)

Mock-infected

Mock-infected/TNF-α

Infected NL4-3Infected NL4-3/TNF-α

0.0

0.6

1.2

1.8

2.4

3.0

3.6

4.2

1 3 5 7 9 12Time Course (day)

Mock-infected

Mock-infected/TNF-α

Infected NL4-3

Infected NL4-3/TNF-α

Figure 6

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Figure 7

0.0

500.0

1000.0

1500.0

2000.0

2500.0

1 2 3 5 7

Time Course (day)

Mock-infected

Mock-infected/TNF-α

Infected VSV (NL4-3)

Infected VSV (NL4-3)/TNF-α

Vira

l Pro

duct

ion

(ng

of p

24)

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1.4X

4.8X

1.5X

2.9X3.2X

5.4X

2.1X

3.8X

1.4X

1.8X

2.6X

4.6X

2.8X

7.8X

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Clade A Clade B Clade C Clade D Clade E Clade F Clade G

Untreated

TNF-α

IL-1β

Luci

fera

se a

ctiv

ity n

orm

aliz

ed in

β-g

alac

tosi

dase

val

ue

Figure 8

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Luci

fera

se A

ctiv

ity (R

LU)

1X

4.4X

7.4X

1.2X

0

30

60

90

120

150

Mock-transfected

LTR-Luc mκB-LTR-Luc NFκB-Luc

Without TNF-α

With TNF-α

Figure 9

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Spontaneous Luciferase Activity (RLU)

Mock-Infected

Table 1 : Susceptibility of Trophoblastic Cell Lines to HIV-1 Infection with Ada-M, HXB-2 and VSV-G Pseudotypes

Luciferase acitivty is expressed in RLU. Results shown are expressed as means ± standard deviations of quadruplate samples and are representative of three experiments.

JAR

JEG-3

0.4 ± 0.07

0.5 ± 0.07

BeWo 0.4 ± 0.03

1.1 ± 0.11

0.5 ± 0.06

0.4 ± 0.02

1.1 ± 0.53

0.3 ± 0.07

0.4 ± 0.03 207.2 ± 28.53

605.9 ± 15.00

281.5 ± 13.73

Ada-Mpseudotype

HXB-2pseudotype

VSV-Gpseudotype

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Gaël Vidricaire, Mélanie R Tardif and Michel J Tremblay and IL-1αpro-inflammatory cytokines TNF-

theinternalization of the human immunodefiency virus type 1 and can be overcome by The low viral production in trophoblastic cells is due to a high endocytic

published online February 25, 2003J. Biol. Chem. 

  10.1074/jbc.M210470200Access the most updated version of this article at doi:

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