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-α and Interleukin-1
2003; Elsevier BV; Volume: 278; Issue: 18 Linguagem: Inglês
10.1074/jbc.m210470200
ISSN1083-351X
AutoresGaël Vidricaire, Mélanie R. Tardif, Michel J. Tremblay,
Tópico(s)HIV Research and Treatment
ResumoMaternal-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-α 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-α 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. 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-α 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-α 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. human immunodeficiency virus type-1 interleukin tumor necrosis factor-α Dulbecco's modified Eagle's medium fetal bovine serum phosphate-buffered saline peripheral blood mononuclear cells vesicular stomatitis virus long terminal repeat relative light units 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. 1Dabs F. Messmate P. Dun D. Lepage P. Newell M.L. Peckham C. Van de Perre P. AIDS. 1993; 7: 1139-1148Crossref PubMed Scopus (154) Google Scholar). In the absence of antiretroviral treatment, the reported risk of vertical transmission lies within the range of 10–39% (1Dabs F. Messmate P. Dun D. Lepage P. Newell M.L. Peckham C. Van de Perre P. AIDS. 1993; 7: 1139-1148Crossref PubMed Scopus (154) Google Scholar, 2Blanche S. Rouzioux C. Moscato M.L. Veber F. Mayaux M.J. Jacomet C. Tricoire J. Deville A. Vial M. Firtion G. De Crepy A. Douard D. Robin M. Courpotin C. Ciragu-Vigneron N. Le Deist F. Griscelli C. the HIV Infection in Newborns French Collaborative Study Group N. Engl. J. Med. 1989; 320: 1643-1648Crossref PubMed Scopus (444) Google Scholar, 3Zachar V. Zacharova V. Fink T. Thomas R.A. King B.R. Ebbesen P. Jones T.B. Goustin A.S. AIDS Res. Hum. Retroviruses. 1999; 15: 1673-1683Crossref PubMed Scopus (27) Google Scholar). 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) (4Thiry L. Sprecher-Goldberger S. Jonckheer T. Levy J. Van de Perre P. Henrivaux P. Cogniaux-LeClerc J. Clumeck N. Lancet. 1985; 2: 891-892Abstract PubMed Scopus (240) Google Scholar, 5Lepage P. Van de Perre P. Carael M. Nsengumuremyi F. Nkurunziza J. Butzler J.P. Sprecher S. Lancet. 1987; 2: 400Abstract PubMed Scopus (98) Google Scholar, 6Willumsen J.F. Newell M.L. Filteau S.M. Coutsoudis A. Dwarika S. York D. Tomkins A.M. Coovadia H.M. AIDS. 2001; 15: 1896-1898Crossref PubMed Scopus (35) Google Scholar). Mathematical modeling has allowed the estimation that 30% of infections occur in utero less than 2 months before birth and 65% at birth (7Rouzioux C. Costagliola D. Burgard M. Blanche S. Mayaux M.J. Griscelli C. Valleron A.J. Am. J. Epidemiol. 1995; 142: 1330-1337Crossref PubMed Scopus (226) Google Scholar). 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 (8Lewis S.H. Reynolds-Kohler C. Fox H.E. Nelson J.A. Lancet. 1990; 335: 565-568Abstract PubMed Scopus (222) Google Scholar, 9Mano H. Chermann J.C. AIDS Res. Hum. Retroviruses. 1991; 7: 83-88Crossref PubMed Scopus (71) Google Scholar, 10Soeiro R. Rubinstein A. Rashbaum W.K. Lyman W.D. J. Infect. Dis. 1992; 166: 699-703Crossref PubMed Scopus (66) Google Scholar, 11Courgnaud V. Laure F. Brossard A. Bignozzi C. Goudeau A. Barin F. Brechot C. AIDS Res. Hum. Retroviruses. 1991; 7: 337-341Crossref PubMed Scopus (109) Google Scholar). 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 (12Lagaye S. Derrien M. Menu E. Coito C. Tresoldi E. Mauclere P. Scarlatti G. Chaouat G. Barre-Sinoussi F. Bomsel M. J. Virol. 2001; 75: 4780-4791Crossref PubMed Scopus (93) Google Scholar). 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 (8Lewis S.H. Reynolds-Kohler C. Fox H.E. Nelson J.A. Lancet. 1990; 335: 565-568Abstract PubMed Scopus (222) Google Scholar, 13De Andreis C. Simoni G. Rossella F. Castagna C. Pesenti E. Porta G. Colucci G. Giuntelli S. Pardi G. Semprini A.E. AIDS. 1996; 10: 711-715Crossref PubMed Scopus (30) Google Scholar, 14Martin A.W. Brady K. 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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 (3Zachar V. Zacharova V. Fink T. Thomas R.A. King B.R. Ebbesen P. Jones T.B. Goustin A.S. AIDS Res. Hum. Retroviruses. 1999; 15: 1673-1683Crossref PubMed Scopus (27) Google Scholar). 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 syncytiotrophoblastsin 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 (19Tscherning-Casper C. Papadogiannakis N. Anvret M. Stolpe L. Lindgren S. Bohlin A.B. Albert J. Fenyo E.M. J. Virol. 1999; 73: 9673-9678Crossref PubMed Google Scholar). 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. 20Vidricaire G. Tremblay M.J. Med. Sci. 2003; (in press)Google Scholar). 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 (9Mano H. Chermann J.C. AIDS Res. Hum. Retroviruses. 1991; 7: 83-88Crossref PubMed Scopus (71) Google Scholar, 21Al-Harthi L. Guilbert L.J. Hoxie J.A. Landay A. AIDS Res. Hum. Retroviruses. 2002; 18: 13-17Crossref PubMed Scopus (22) Google Scholar, 22David F.J. Autran B. Tran H.C. Menu E. Raphael M. Debre P. Hsi B.L. Wegman T.G. Barre-Sinoussi F. Chaouat G. Clin. Exp. Immunol. 1992; 88: 10-16Crossref PubMed Scopus (56) Google Scholar, 23David F.J. Tran H.C. Serpente N. Autran B. Vaquero C. Djian V. Menu E. Barre-Sinoussi F. Chaouat G. Virology. 1995; 208: 784-788Crossref PubMed Scopus (29) Google Scholar, 24Mognetti B. Moussa M. Croitoru J. Menu E. Dormont D. Roques P. Chaouat G. Clin. Exp. Immunol. 2000; 119: 486-492Crossref PubMed Scopus (55) Google Scholar). 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-β, interferon-γ and TNF-α), 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. 25Robertson S.A. Seamark R.F. Guilbert L.J. Wegmann T.G. Crit. Rev. Immunol. 1994; 14: 239-292Crossref PubMed Google Scholar, 26Saito S. J. Reprod. Immunol. 2000; 47: 87-103Crossref PubMed Scopus (315) Google Scholar, 27Norwitz E.R. Schust D.J. Fisher S.J. N. Engl. J. Med. 2001; 345: 1400-1408Crossref PubMed Scopus (852) Google Scholar). 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 expressionin vitro (reviewed in Refs. 20Vidricaire G. Tremblay M.J. Med. Sci. 2003; (in press)Google Scholar and 28Poli G. Fauci A.S. Human Cytokines: Their Role in Disease and Therapy. Blackwell Sciences Ltd., USA1995: 421-449Google Scholar). 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-γ, IL-1α, IL-1β, 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-β, and TNF-α), only two cytokines, TNF-α and IL-1, were found to strongly activate virus transcription (29Vidricaire, G., Tremblay, M. J., 10th Conference on Retroviruses and Opportunistic Infections, 2003, 370, Foundation for Retrovirology and Human Health, Alexandria, VA, Boston, MA, February 2003.Google Scholar). 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 (30Canki M. Thai J.N. Chao W. Ghorpade A. Potash M.J. Volsky D.J. J. Virol. 2001; 75: 7925-7933Crossref PubMed Scopus (105) Google Scholar). 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-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 transmission of HIV-1 to the fetus during gestation. 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) (31Lahm H.W. Stein S. J. Chromatogr. 1985; 326: 357-361Crossref PubMed Scopus (107) Google Scholar). 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 (32Azocar J. Essex M. Cancer Res. 1979; 39: 3388-3391PubMed Google Scholar, 33Connor R.I. Chen B.K. Choe S. Landau N.R. Virology. 1995; 206: 935-944Crossref PubMed Scopus (1082) Google Scholar). 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 (34Yee J.K. Miyanohara A. LaPorte P. Bouic K. Burns J.C. Friedmann T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9564-9568Crossref PubMed Scopus (442) Google Scholar). 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). These molecular constructs contain the luciferase reporter gene under the control of wild-type (GGGACTTTCC) or NF-κB-mutated (CTC ACTTTCC) HIV-1HXB2 LTR (453 to +80) (35Henderson A.J. Zou X. Calame K.L. J. Virol. 1995; 69: 5337-5344Crossref PubMed Google Scholar). The molecular construct pNF-κB-Luc contains five (5Lepage P. Van de Perre P. Carael M. Nsengumuremyi F. Nkurunziza J. Butzler J.P. Sprecher S. Lancet. 1987; 2: 400Abstract PubMed Scopus (98) Google Scholar) 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 subtypes A to G driving the firefly luciferase reporter gene (obtained through the AIDS Repository Reagent Program) (36Jeeninga R.E. Hoogenkamp M. Armand-Ugon M. de Baar M. Verhoef K. Berkhout B. J. Virol. 2000; 74: 3740-3751Crossref PubMed Scopus (240) Google Scholar). Viruses were produced by calcium phosphate transfection of 293T cells, as described previously (37Chen B.K. Saksela K. Andino R. Baltimore D. J. Virol. 1994; 68: 654-660Crossref PubMed Google Scholar, 38Fortin J.F. Cantin R. Lamontagne G. Tremblay M. J. Virol. 1997; 71: 3588-3596Crossref PubMed Google Scholar). 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 (39Bounou S. Leclerc J.E. Tremblay M.J. J. Virol. 2002; 76: 1004-1014Crossref PubMed Scopus (163) Google Scholar). 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 (39Bounou S. Leclerc J.E. Tremblay M.J. J. Virol. 2002; 76: 1004-1014Crossref PubMed Scopus (163) Google Scholar). 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 (39Bounou S. Leclerc J.E. Tremblay M.J. J. Virol. 2002; 76: 1004-1014Crossref PubMed Scopus (163) Google Scholar). 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 × 103cells 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) and interleukin (IL)-1α or IL-1β (NCI, National Institutes of Health, Bethesda). After an 8- to 72-h incubation period, luciferase activity was monitored in cell lysates as described previously (40Barbeau B. Bernier R. Dumais N. Briand G. Olivier M. Faure R. Posner B.I. Tremblay M. J. Biol. Chem. 1997; 272: 12968-12977Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). 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-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 × 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. 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 β-galactosidase vector (pActin-β-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-α 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. β-Galactosidase assays were performed using the Galacto LightTM chemiluminescent reporter assay for β-galactosidase (Tropix, Bedford, MA). Given the reported low susceptibility of trophoblastic cells to productive HIV-1 infection (22David F.J. Autran B. Tran H.C. Menu E. Raphael M. Debre P. Hsi B.L. Wegman T.G. Barre-Sinoussi F. Chaouat G. Clin. Exp. Immunol. 1992; 88: 10-16Crossref PubMed Scopus (56) Google Scholar, 23David F.J. Tran H.C. Serpente N. Autran B. Vaquero C. Djian V. Menu E. Barre-Sinoussi F. Chaouat G. Virology. 1995; 208: 784-788Crossref PubMed Scopus (29) Google Scholar, 41Zachar V. Spire B. Hirsch I. Chermann J.C. Ebbesen P. J. Virol. 1991; 65: 2102-2107Crossref PubMed Google Scholar), 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 (30Canki M. Thai J.N. Chao W. Ghorpade A. Potash M.J. Volsky D.J. J. Virol. 2001; 75: 7925-7933Crossref PubMed Scopus (105) Google Scholar) 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 trop
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