The Low Viral Production in Trophoblastic Cells Is Due to a High Endocytic Internalization of the Human Immunodeficiency Virus Type 1 and Can Be Overcome by the Pro-inflammatory Cytokines Tumor Necrosis Factor-alpha and Interleukin-1*

Gaël VidricaireDagger§, Mélanie R. Tardif§, and Michel J. Tremblay

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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-alpha 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta , interferon-gamma and TNF-alpha ), 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-gamma , IL-1alpha , IL-1beta , 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-beta , and TNF-alpha ), only two cytokines, TNF-alpha 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.

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-alpha 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-alpha 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
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INTRODUCTION
MATERIALS AND METHODS
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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-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 pmkappa 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-kappa B-mutated (CTCACTTTCC) HIV-1HXB2 LTR (453 to +80) (35). The molecular construct pNF-kappa B-Luc contains five (5) consensus sequences of NF-kappa 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).

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-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 × 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)-alpha (R&D Systems, Minneapolis, MN) and interleukin (IL)-1alpha or IL-1beta (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-alpha 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.

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 beta -galactosidase vector (pActin-beta -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-alpha 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. beta -Galactosidase assays were performed using the Galacto LightTM chemiluminescent reporter assay for beta -galactosidase (Tropix, Bedford, MA).

    RESULTS
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MATERIALS AND METHODS
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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.


                              
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Table I
Susceptibility of trophoblastic cell lines to HIV-1 infection with Ada-M, HXB-2, and VSV-G pseudotypes
Luciferase activity is expressed in RLU. Results shown are expressed as means ± S.D. of quadruplicate samples and are representative of three experiments.

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.


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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.

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.


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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.

HIV-1 LTR-driven Reporter Gene Activity Is Increased in Trophoblastic Cells by TNF-alpha , IL-1alpha , and IL-1beta -- 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-alpha , IL-1alpha , and IL-1beta 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-alpha and IL-1beta (Fig. 3). For example, incubation of Ada-M-infected JAR cells with increasing doses of TNF-alpha and IL-1beta 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-1alpha (data not shown). Further studies revealed that the positive effect of TNF-alpha , IL-1alpha , and IL-1beta 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-alpha , IL-1alpha , and IL-1beta 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-alpha (i.e. 8.2-fold increase with the highest dose of TNF-alpha ). Exposure of BeWo cells to IL-1alpha and IL-1beta produced a 4-fold increase in HIV-1 LTR activity. Similar data were obtained when using JEG-3 cells (data not shown).


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Fig. 3.   HIV-1 transcriptional activity is increased upon treatment of trophoblasts with TNF-alpha and IL-1beta . 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-alpha (A and C) or IL-1beta (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.


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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-alpha (A), IL-1alpha (B), or IL-1beta (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.


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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-alpha (A), IL-1alpha (B), or IL-beta (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.

Production of Fully Infectious HIV-1 Particles by Trophoblastic Cells Is Only Seen Following Treatment with TNF-alpha 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-alpha . 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-alpha (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-alpha -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-alpha . 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-alpha .


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Fig. 6.   Production of fully infectious HIV-1 particles is seen when virus-infected trophoblasts are treated with TNF-alpha 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-alpha 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.

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-alpha . 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-alpha (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-alpha 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.

Different HIV-1 LTR Subtypes Are Transactivated by TNF-alpha and IL-1beta 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-alpha and IL-1beta . As shown in Fig. 8, virus transcription was triggered by both TNF-alpha and IL-1beta 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-alpha and IL-1beta 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 beta -galactosidase vector. Cells were next either left untreated or treated with TNF-alpha or IL-1beta and were lysed at 8 h post-stimulation. HIV-1-encoded luciferase activity and actin-dependent beta -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 beta -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.

Cytokine-dependent Up-regulation of HIV-1 LTR Activity in Trophoblasts Is Mediated via NF-kappa B-- Finally, we investigated the molecular events leading to TNF-alpha -induced transactivation of HIV-1 by transiently transfecting the JAR cell line with various expression vectors before exposure to TNF-alpha . As depicted in Fig. 9, TNF-alpha 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-kappa B-binding sites within the HIV-1HXB2 LTR domain. The involvement of NF-kappa 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-alpha to JAR cells that were transfected with a kappa B-driven reporter gene vector.


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Fig. 9.   Cytokine-dependent transactivation of HIV-1 LTR in trophoblasts is mediated via NF-kappa B. JAR cells were transiently transfected with pLTR-Luc, pmkappa BLTR-Luc, or pNF kappa B-Luc and were either left untreated or treated with TNF-alpha . 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

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-alpha , IL-1alpha , and IL-1beta 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-alpha and IL-1beta 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.

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-alpha and/or IL-1. These newly released virions, as we observed when HIV-1-infected trophoblasts were treated with TNF-alpha 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-alpha 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-alpha , IL-1beta , 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-alpha and IL-1beta 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-alpha and IL-6 by macrophages (52). Based on these published data together with the present findings, it can be proposed that TNF-alpha 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-alpha 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-alpha 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-1alpha and IL-1beta , 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-alpha . 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-alpha 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-kappa B. The TNF-alpha -dependent signaling pathway has been extensively studied in lymphocytic and monocytic cells, and NF-kappa 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).

The roles played by TNF-alpha 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-alpha 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-alpha 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-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-alpha 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-alpha 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.

    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.

Dagger 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-alpha , tumor necrosis factor-alpha ; 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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ABSTRACT
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RESULTS
DISCUSSION
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