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A human immunodeficiency virus Env inducible transcription system to examine consequences of gp120 expression
Journal of Virological Methods 98 (2001) 145– 151 www.elsevier.com/locate/jviromet
A human immunodeficiency virus Env inducible transcription system to examine consequences of gp120 expression Ve´ronique Joliot a,b,1, Caroline Goujon a, Julie Dumonceaux a,2, Agne´s Renard a,3, Pascale Briand a, Uriel Hazan a,b,* a
INSERM Unite´ 380, Laboratoire de Ge´ne´tique et Pathologie Expe´rimentales, Institut Cochin de Ge´ne´tique Mole´culaire, 22 Rue Me´chain, 75014, Paris, France b Uni6ersite´ Paris 7 -Denis Diderot, UFR de Biochimie, 75005 Paris, France Received 9 May 2001; received in revised form 20 July 2001; accepted 23 July 2001
Abstract According to several studies, the HIV-1 envelope gp120 protein and the co-receptor CXCR4 play an essential role in HIV-1 induced cell toxicity. Characterisation of the CD4-independent m7NDK isolate provided the opportunity of studying the effects of direct interactions between m7NDK gp120 and CXCR4. Therefore, an inducible expression system was designed enabling synthesis of HIV-1 Env proteins upon doxycycline induction. Analysis of the expression of the en6 gene of the m7NDK HIV-1 isolate revealed, unexpectedly, that even long-term expression of m7NDK gp120 did not result in cytotoxycity in CXCR4-positive or -negative cell lines. This is the first report of a CD4-independent HIV-1-protein inducible expression regulated through the Tet-On system and by an alternative splicing. Env inducible expression cell lines could constitute a useful cellular tool to undertake analysis of HIV Env protein expression. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Inducible expression system; gp120; CD4-independant
1. Introduction * Corresponding author. Tel.: + 33-1-405-16455; fax: + 331-405-16407. E-mail address: [email protected] (U. Hazan). 1 Present address: CNRS UMR146, Laboratoire de Re´gulations Cellulaires et Oncoge´ne`se, Institut Curie, 91405, Orsay, France. 2 Present address: Microbiology and Immunology Department, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. 3 Present address: IBIODEM, Ge´nopoˆle Industries, 4 Rue Pierre Fontaine, 91000 Evry, France.
The human immunodeficiency virus (HIV) gp120 envelope (Env) glycoproteins mediate interaction with cellular receptors thus resulting in viral and cellular membrane fusion (Berger et al., 1999). The first step implies CD4 binding (Sattentau et al., 1993) which induces conformational changes in the gp120 allowing efficient interactions with the co-receptor (for review see (Choe et al., 1998; Sattentau, 1998)), most members being
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chemokine receptors and related orphans and CXCR4 and CCR5 being the principal co-receptors identified in vivo (Berger et al., 1999; Clapham et al., 1999). In vitro, a few CD4-independent HIV isolates have been characterised (Clapham et al., 1992; Dumonceaux et al., 1998; Endres et al., 1996; Hoxie et al., 1998; Kolchinsky et al., 1999; Reeves et al., 1999, 1997), including the first CD4-independent HIV-1 isolate, the m7NDK, characterised in our laboratory (Dumonceaux et al., 1998). As these viruses are CD4independent, direct interactions between the gp120 and the co-receptor might allow virus entry (Dumonceaux et al., 1998; Hoxie et al., 1998; Potempa et al., 1997). Some studies undertaken with CD4-dependent virus highlighted that the gp120 might induce intracellular signaling (Briant et al., 1998; Popik et al., 1998; Popik and Pitha, 1996) likely to be involved in HIV-1-induced cell toxicity (Ameisen, 1998; Ikuta et al., 1997). Moreover, it has been suggested that gp120/CXCR4 rather than gp120/ CD4 interactions induce an essential CXCR4 signaling which may be correlated with the triggering of cell death (Gabuzda and Wang, 2000). All these studies were carried out with CD4-dependent envelope proteins in cells expressing normal CD4 or truncated CD4 to avoid CD4 signaling. The discovery of CD4-independent HIV-1 isolates provided the opportunity for studying the putative effects of direct interaction between gp120 and CXCR4. Constitutive expression of the m7NDK en6 gene was assessed using cell lines which do not express CD4 but which express CXCR4 (HeLa and Cos7 cells) or not (U373MG). Cellular clones exhibiting constitutive expression of the wtNDK en6 gene were obtained from all the cell lines used. Conversely, for the m7NDK en6 gene all the cellular clones derived from HeLa or COS7 cells were negative for Env expression and only one clone from the CXCR4 negative cell line (U373MG) exhibited a constitutive expression of the m7NDK en6 gene (Joliot et al. Unpublished data). These preliminary results thus suggested that cells could not survive concomitant expression of CD4-independent envelope and CXCR4 during positive selection of drug-resistant cell clones.
An Env inducible expression system was therefore developed to allow stable expression of wtNDK and m7NDK en6 genes. Finally, even though no cytotoxic effect or critical alteration on cell proliferation could be demonstrated with the m7NDK gp120, Env inducible expression cell lines might constitute a useful cellular tool to carry out analysis of HIV Env protein expression.
2. Materials and methods
2.1. Cell lines and expression system The CXCR4 U373MG astrocytic human cell line was a gift from Dr J.-L. Virelizier, Institut Pasteur, Paris. The rtTa–HeLa cell line stably expresses rtTa (Clontech) and is maintained under G148 selection (500 mg/mL, Life Technologies). The U373 cell line was transfected with the rtTa constitutive expression plasmid (Clontech). Cellular clones presenting a high rtTa expression level were selected after G418 selection (500 mg/mL, Life Technologies, rtTa-U373). U373MGLTRLuc reporter cell lines containing the luciferase gene downstream of the Tat-inducible HIV-1 promoter and expressing CXCR4 and either CD4 (X31CD4) or not (X31) have been described (Dumonceaux et al., 1999). They were grown with 500 mg/mL G418 (Life Technologies) for X31 cells and G418 and hygromycin B (150 mg/mL; Calbiochem) for X31CD4 cells. All cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal calf serum, antibiotics and glutamine (Life Technologies).
2.2. Establishment of En6 inducible cellular clones 2.2.1. Construction of expression 6ectors A puromycin resistance gene under the control of the phosphoglycerate kinase promoter (Adra et al., 1987) was introduced into the pTRE plasmid (Gossen and Bujard, 1992). Cloning sites were redesigned to subclone the common HIV-1 splice donor site together with m7NDK env-containing regions (Dumonceaux et al., 1998) downstream of the inducible promoter using EcoRI/XhoI enzymes. Briefly, the XhoI unique site of the pTRE
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plasmid was filled-in using Keenow enzyme (Biolabs) and an XhoI linker (Biolabs) was inserted in the XbaI restriction site located in the multicloning site (Gossen and Bujard, 1992). The m7NDK env-containing regions were then eliminated using NotI/XhoI enzymes and exchanged with wtNDK counterpart regions (Dumonceaux et al., 1998). The resulting vectors could express Env, Tat, Rev and Vpu proteins under doxycycline induction and through alternative spliced between the common Splice donor site from the promoter region and gene specific acceptor sites.
2.2.2. Transfection experiments Transfection were obtained using the calcium phosphate coprecipitation method (Chen and Okayama, 1987) for rtTA– HeLa and the Transfast System (Qiagen) according to the manufacturer’s recommendations for U373-MG cell lines, respectively. 2.2.3. Selection of stable clones Cells were transfected with 10 mg of either wtgp120 or m7gp120 inducible expression plasmids. After transfection, cells were trysinized, diluted and were grown under puromycin selection (Calbiochem, 4 mg/mL). Individual clones were isolated after cylinder cloning.
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drogenase (GAPDH) probe (GenBank Access No: XM-006959) were used as internal controls.
2.3.2. Western blot experiments Western blot experiments were carried out on cell lysates obtained according to the Laemmli procedure (Cleveland et al., 1977). The gp120 was revealed with the monoclonal anti-gp120 Ab D7324 (Aalto) which was raised against a C-terminal peptide of the gp120. Chemoluminescence (ECL, Amersham) was monitored by autoradiography after incubation with an HRPO-coupled anti-sheep Ab (Dako). 2.3.3. Fusion test Envelope functionality was assayed by a fusion test as described previously (Dumonceaux et al., 1998). Briefly, Env expressing cells were mixed (ratio 1:1) with U373MGLTRLuc reporter cell lines expressing CXCR4 and either CD4 (X31CD4) or not (X31) (Dumonceaux et al., 1999). Fusion efficiency was analyzed through Luciferase activity of cell lysates at different times upon doxycycline induction. Luciferase tests were performed on a Lumat-LB9507 luminometer (EGGBerthold).
3. Results
2.3. Analysis of En6 expression 2.3.1. Northern blot experiments After doxycycline induction, cells were collected after various incubation periods as indicated and were used for RNA extraction. Total cellular RNA extraction and purification was carried out using an RNA B™ isolation system (Bioprobe) following manufacturer’s protocol. RNA was extracted from induced cells as indicated, and 10 mg of each preparation were denatured with formaldehyde and size-fractionated by electrophoresis on a 1% agarose gel. The RNAs were then transferred to a hybridization transfer membrane and hybridized with a 32P-labelled probe complementary to the leader region upstream of the common splice donor site, the 5% part of the en6 gene and exon domains of tat, rev and vpu. Hybridizations using a human glyceraldehyde-3-phosphate dehy-
The Tet-On inducible system (Clontech) in which the rtTa transactivator is functional for activating transcription only when doxycycline is added was used thereafter. Inducible vectors driving the expression of either m7NDK gp120 or wtNDK gp120 proteins were then transfected in both rtTa–HeLa and the rtTa-U373 cell lines. Cellular clones expressing fusion-competent envelope proteins upon doxycycline induction were isolated. Results are only reported from rtTa– HeLa cellular clones since rtTa-U373 clones gave similar results (data not shown). Envelope functionality was assayed by a fusion test after coculture with U373MGLTRLuc reporter cell lines expressing CXCR4 and either CD4 (X31CD4) or not (X31) (Dumonceaux et al., 1999). Fusion efficiency was analyzed by Luciferase activity of cell lysates at different times upon doxycycline
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induction. Results from two representative rtTa– HeLa clones expressing either wtNDK (H-wtNDK) or m7NDK (H-m7NDK) en6 genes are shown in Fig. 1. With the CD4-positive indicator X31CD4 cell line (panel A), the Luciferase activity was clearly detected from 24 h after addition of doxycycline and then increased to reach an almost constant level after 72 h. Activities increased up to 40 fold for H-wtNDK cells and up to 35 fold for H-m7NDK ones. With the CD4negative indicator X31 cell line (panel B), Luciferase activity was only detected after coculture with H-m7NDK cells and followed a similar timecourse, up to a 9-fold increase. As previously described, even if the m7NDK gp120 exhibits a CD4-independent tropism, cell fusion is more effi-
Fig. 1. Kinetic analysis of Env expression by coculture experiments. CD4-dependent (H-wtNDK) or CD4-independent (Hm7NDK) Env expression cell lines were induced by addition of doxycycline. Cell fusion efficiency was estimated at different times after induction and after cocultures either with X31CD4 (U373-MG, LTR-Luc, CXCR4+, CD4+) (Panel A) or with X31 (U373-MG, LTR-Luc, CXCR4+) (Panel B) followed by Luciferase measurements.
cient in the presence of CD4 (Dumonceaux et al., 1998). It should be noted that during this short time-course expression, the CD4-independent phenotype was clearly maintained, as expected (Fig. 1B). The time-course induction of envelope expression was also analyzed at RNA and protein levels. Northern blot hybridization experiments (Fig. 2A) were carried out with total RNA. Three RNAs were detected, the larger one (3.4 kb) corresponding to the en6 mRNA and smaller species (2.6 and 2.0 kb) corresponding to auxiliary genes mRNAs (Fig. 2A). The en6 mRNA was detectable from 16 h after induction and its amount regularly increased to become maximal after 96 h, which was confirmed after monitoring of related GAPDH mRNA levels (Fig. 2A). Western blot experiments (Fig. 2B), were carried out on cell lysates using the monoclonal anti-gp120 Ab D7324. In our experiments, only the gp120 env protein was detected, which suggests that the epitope does not present the same conformation in the gp160 env precursor. The gp120 protein was slightly detectable 16 h after induction and exhibited a regular increase up to 96 h to reach an almost constant level later on. These different time-course analyses demonstrated clearly that gp120 expression could be controlled by the addition of doxycycline. Long-term induction (up to 3 months) was then carried out and Env expression was regularly assayed for both H-m7NDK and H-wtNDK cells. It should be noted that the level reached after long-term induction was similar to that of constitutively expressed wNDK gp120 (data not shown). During these long-term inductions, cells proliferated normally, autofusion events in the H-m7NDK cultures were observed and the envelope gene was always expressed (Fig. 3). Moreover, the CD4-independent phenotype remained unaltered (Fig. 3).
4. Discussion Inducible HIV Env expressing cell lines, tightly controlled through doxycycline induction were engineered and characterised. Indeed, no basal Env-
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Fig. 2. Time-course analysis of HIV or GAPDH specific RNA expression (A) and Western-Blot analysis of gp120 kinetics of expression (B). (A) Total RNA were isolated either from the H-m7NDK or the H-wtNDK cell lines, at different times after doxycycline induction (lane 1 =0 h; 2 =4 h; 3= 8 h; 4= 16 h; 5 = 24 h; 6 =48 h; 7 = 72 h; 8 =96 h and 9 =1 week). Membranes were hybridized either with an HIV-1 en6, tat, re6, 6pu or GAPDH-specific labeled probe, as indicated. These results are representative of three different experiments. (B) Cell lysates were prepared at different times after doxycycline induction (lane 1 =0 h; 2 =16 h; 3= 24 h; 4 = 48 h; 5=72 h; 6 =96 h; 7 = 1 week) from CD4-dependent (HwtNDK) or CD4-independent (H-m7NDK) Env-expressing cell lines, as indicated. Env inducible expressed gp120 were analyzed after incubation with a monoclonal anti-gp120 (D7324). These results are representative of three different experiments.
dependent fusion could be observed before addition of the inducer (Fig. 1 and not shown). The maximum expression level was obtained within 72–96 h thus suggesting a progressive expression of the en6 gene. Moreover, FACS analysis of gp120 expression using a polyclonal human antiserum did not indicate any difference between both m7gp120 and wtgp120. This expression level remained unchanged after an induction period of 3 months (data not shown). A similar time-course for Env proteins inducible expression has been described previously (Cao et al., 1996). This delayed expression of Env proteins seem to result from post-transcriptional events since in the same rtTA-cell lines it was observed that the doxycycline-inducible luciferase reporter construct (Gossen and Bujard, 1992) reached a maximum expression level as soon as 18 h (data not shown). Nevertheless, the delayed expression of Env proteins could not be correlated with a preferential early expression of auxiliary gene mRNAs, as could have been expected (Cullen, 1993).
The first concern was to assay the effects of concomitant expression of a CD4-independent envelope protein and CXCR4 and to determine if direct gp120/CXCR4 interaction could affect cell survival. During time-course experiments no critical alteration of cell proliferation could be observed. H-wtNDK and H-m7NDK cell cultures were carried out similarly at low density to avoid, as much as possible, autofusion in the H-m7NDK cultures. Moreover, the CD4-independent phenotype remained unaltered (Fig. 3), even though it could have been hypothesized that cell survival might have been associated with an extinction of the expression of either CD4-independent envelope or of CXCR4 genes (Valente et al., 2001). Indeed, FACS analysis of both m7NDK and wtNDK gp120s and CXCR4 expression did not indicate any modification during the whole period (data not shown). However, the results with Env inducible expression cell lines contradict our initial findings that clones which expressed m7NDK Env besides
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References
Fig. 3. Analysis of Env expression by coculture experiments after long-term induction. CD4-dependent (H-wtNDK) or CD4-independent (H-m7NDK) Env inducible expression cell lines were induced by addition of doxycycline either for several days or several months. Cell fusion efficiencies were estimated at different times after induction and after cocultures either with X31CD4 (U373-MG, LTR-Luc, CXCR4+, CD4+) or with X31 (U373-MG, LTR-Luc, CXCR4+) followed by Luciferase measurements. Only two representative time-course results are shown for each of the inducible cell lines.
CXCR4 were not viable. Finally, we described a CD4-independent HIV-1-Env expression system in which transcription is directed from an inducible heterologous promoter allowing alternative splicing. Env inducible expression cell lines could constitute a useful cellular tool for carrying with analysis of HIV Env protein expression.
Acknowledgements We are grateful for the technical support of C. Montigny and L. Bougne`res. N. Boord edited the English. J.D. hold a fellowship from Ministe`re de l’Education Nationale, de l’Enseignement Supe´ neur et de la Recherche. A.R. was supported by a grant from Fondation des Treilles. This work was supported by grants from Agence Nationale de la Recherche contre le SIDA (ANRS), ‘Ensemble Contre le SIDA’ AO11, by Fondation Pour la Recherche Me´ dicale and by ‘Lutte AntiSida’ AO2 of the University of Paris 7-Denis Diderot.
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A human immunodeficiency virus Env inducible transcription system to examine consequences of gp120 expression Ve´ronique Joliot a,b,1, Caroline Goujon a, Julie Dumonceaux a,2, Agne´s Renard a,3, Pascale Briand a, Uriel Hazan a,b,* a
INSERM Unite´ 380, Laboratoire de Ge´ne´tique et Pathologie Expe´rimentales, Institut Cochin de Ge´ne´tique Mole´culaire, 22 Rue Me´chain, 75014, Paris, France b Uni6ersite´ Paris 7 -Denis Diderot, UFR de Biochimie, 75005 Paris, France Received 9 May 2001; received in revised form 20 July 2001; accepted 23 July 2001
Abstract According to several studies, the HIV-1 envelope gp120 protein and the co-receptor CXCR4 play an essential role in HIV-1 induced cell toxicity. Characterisation of the CD4-independent m7NDK isolate provided the opportunity of studying the effects of direct interactions between m7NDK gp120 and CXCR4. Therefore, an inducible expression system was designed enabling synthesis of HIV-1 Env proteins upon doxycycline induction. Analysis of the expression of the en6 gene of the m7NDK HIV-1 isolate revealed, unexpectedly, that even long-term expression of m7NDK gp120 did not result in cytotoxycity in CXCR4-positive or -negative cell lines. This is the first report of a CD4-independent HIV-1-protein inducible expression regulated through the Tet-On system and by an alternative splicing. Env inducible expression cell lines could constitute a useful cellular tool to undertake analysis of HIV Env protein expression. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Inducible expression system; gp120; CD4-independant
1. Introduction * Corresponding author. Tel.: + 33-1-405-16455; fax: + 331-405-16407. E-mail address: [email protected] (U. Hazan). 1 Present address: CNRS UMR146, Laboratoire de Re´gulations Cellulaires et Oncoge´ne`se, Institut Curie, 91405, Orsay, France. 2 Present address: Microbiology and Immunology Department, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. 3 Present address: IBIODEM, Ge´nopoˆle Industries, 4 Rue Pierre Fontaine, 91000 Evry, France.
The human immunodeficiency virus (HIV) gp120 envelope (Env) glycoproteins mediate interaction with cellular receptors thus resulting in viral and cellular membrane fusion (Berger et al., 1999). The first step implies CD4 binding (Sattentau et al., 1993) which induces conformational changes in the gp120 allowing efficient interactions with the co-receptor (for review see (Choe et al., 1998; Sattentau, 1998)), most members being
0166-0934/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 3 7 3 - 1
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chemokine receptors and related orphans and CXCR4 and CCR5 being the principal co-receptors identified in vivo (Berger et al., 1999; Clapham et al., 1999). In vitro, a few CD4-independent HIV isolates have been characterised (Clapham et al., 1992; Dumonceaux et al., 1998; Endres et al., 1996; Hoxie et al., 1998; Kolchinsky et al., 1999; Reeves et al., 1999, 1997), including the first CD4-independent HIV-1 isolate, the m7NDK, characterised in our laboratory (Dumonceaux et al., 1998). As these viruses are CD4independent, direct interactions between the gp120 and the co-receptor might allow virus entry (Dumonceaux et al., 1998; Hoxie et al., 1998; Potempa et al., 1997). Some studies undertaken with CD4-dependent virus highlighted that the gp120 might induce intracellular signaling (Briant et al., 1998; Popik et al., 1998; Popik and Pitha, 1996) likely to be involved in HIV-1-induced cell toxicity (Ameisen, 1998; Ikuta et al., 1997). Moreover, it has been suggested that gp120/CXCR4 rather than gp120/ CD4 interactions induce an essential CXCR4 signaling which may be correlated with the triggering of cell death (Gabuzda and Wang, 2000). All these studies were carried out with CD4-dependent envelope proteins in cells expressing normal CD4 or truncated CD4 to avoid CD4 signaling. The discovery of CD4-independent HIV-1 isolates provided the opportunity for studying the putative effects of direct interaction between gp120 and CXCR4. Constitutive expression of the m7NDK en6 gene was assessed using cell lines which do not express CD4 but which express CXCR4 (HeLa and Cos7 cells) or not (U373MG). Cellular clones exhibiting constitutive expression of the wtNDK en6 gene were obtained from all the cell lines used. Conversely, for the m7NDK en6 gene all the cellular clones derived from HeLa or COS7 cells were negative for Env expression and only one clone from the CXCR4 negative cell line (U373MG) exhibited a constitutive expression of the m7NDK en6 gene (Joliot et al. Unpublished data). These preliminary results thus suggested that cells could not survive concomitant expression of CD4-independent envelope and CXCR4 during positive selection of drug-resistant cell clones.
An Env inducible expression system was therefore developed to allow stable expression of wtNDK and m7NDK en6 genes. Finally, even though no cytotoxic effect or critical alteration on cell proliferation could be demonstrated with the m7NDK gp120, Env inducible expression cell lines might constitute a useful cellular tool to carry out analysis of HIV Env protein expression.
2. Materials and methods
2.1. Cell lines and expression system The CXCR4 U373MG astrocytic human cell line was a gift from Dr J.-L. Virelizier, Institut Pasteur, Paris. The rtTa–HeLa cell line stably expresses rtTa (Clontech) and is maintained under G148 selection (500 mg/mL, Life Technologies). The U373 cell line was transfected with the rtTa constitutive expression plasmid (Clontech). Cellular clones presenting a high rtTa expression level were selected after G418 selection (500 mg/mL, Life Technologies, rtTa-U373). U373MGLTRLuc reporter cell lines containing the luciferase gene downstream of the Tat-inducible HIV-1 promoter and expressing CXCR4 and either CD4 (X31CD4) or not (X31) have been described (Dumonceaux et al., 1999). They were grown with 500 mg/mL G418 (Life Technologies) for X31 cells and G418 and hygromycin B (150 mg/mL; Calbiochem) for X31CD4 cells. All cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal calf serum, antibiotics and glutamine (Life Technologies).
2.2. Establishment of En6 inducible cellular clones 2.2.1. Construction of expression 6ectors A puromycin resistance gene under the control of the phosphoglycerate kinase promoter (Adra et al., 1987) was introduced into the pTRE plasmid (Gossen and Bujard, 1992). Cloning sites were redesigned to subclone the common HIV-1 splice donor site together with m7NDK env-containing regions (Dumonceaux et al., 1998) downstream of the inducible promoter using EcoRI/XhoI enzymes. Briefly, the XhoI unique site of the pTRE
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plasmid was filled-in using Keenow enzyme (Biolabs) and an XhoI linker (Biolabs) was inserted in the XbaI restriction site located in the multicloning site (Gossen and Bujard, 1992). The m7NDK env-containing regions were then eliminated using NotI/XhoI enzymes and exchanged with wtNDK counterpart regions (Dumonceaux et al., 1998). The resulting vectors could express Env, Tat, Rev and Vpu proteins under doxycycline induction and through alternative spliced between the common Splice donor site from the promoter region and gene specific acceptor sites.
2.2.2. Transfection experiments Transfection were obtained using the calcium phosphate coprecipitation method (Chen and Okayama, 1987) for rtTA– HeLa and the Transfast System (Qiagen) according to the manufacturer’s recommendations for U373-MG cell lines, respectively. 2.2.3. Selection of stable clones Cells were transfected with 10 mg of either wtgp120 or m7gp120 inducible expression plasmids. After transfection, cells were trysinized, diluted and were grown under puromycin selection (Calbiochem, 4 mg/mL). Individual clones were isolated after cylinder cloning.
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drogenase (GAPDH) probe (GenBank Access No: XM-006959) were used as internal controls.
2.3.2. Western blot experiments Western blot experiments were carried out on cell lysates obtained according to the Laemmli procedure (Cleveland et al., 1977). The gp120 was revealed with the monoclonal anti-gp120 Ab D7324 (Aalto) which was raised against a C-terminal peptide of the gp120. Chemoluminescence (ECL, Amersham) was monitored by autoradiography after incubation with an HRPO-coupled anti-sheep Ab (Dako). 2.3.3. Fusion test Envelope functionality was assayed by a fusion test as described previously (Dumonceaux et al., 1998). Briefly, Env expressing cells were mixed (ratio 1:1) with U373MGLTRLuc reporter cell lines expressing CXCR4 and either CD4 (X31CD4) or not (X31) (Dumonceaux et al., 1999). Fusion efficiency was analyzed through Luciferase activity of cell lysates at different times upon doxycycline induction. Luciferase tests were performed on a Lumat-LB9507 luminometer (EGGBerthold).
3. Results
2.3. Analysis of En6 expression 2.3.1. Northern blot experiments After doxycycline induction, cells were collected after various incubation periods as indicated and were used for RNA extraction. Total cellular RNA extraction and purification was carried out using an RNA B™ isolation system (Bioprobe) following manufacturer’s protocol. RNA was extracted from induced cells as indicated, and 10 mg of each preparation were denatured with formaldehyde and size-fractionated by electrophoresis on a 1% agarose gel. The RNAs were then transferred to a hybridization transfer membrane and hybridized with a 32P-labelled probe complementary to the leader region upstream of the common splice donor site, the 5% part of the en6 gene and exon domains of tat, rev and vpu. Hybridizations using a human glyceraldehyde-3-phosphate dehy-
The Tet-On inducible system (Clontech) in which the rtTa transactivator is functional for activating transcription only when doxycycline is added was used thereafter. Inducible vectors driving the expression of either m7NDK gp120 or wtNDK gp120 proteins were then transfected in both rtTa–HeLa and the rtTa-U373 cell lines. Cellular clones expressing fusion-competent envelope proteins upon doxycycline induction were isolated. Results are only reported from rtTa– HeLa cellular clones since rtTa-U373 clones gave similar results (data not shown). Envelope functionality was assayed by a fusion test after coculture with U373MGLTRLuc reporter cell lines expressing CXCR4 and either CD4 (X31CD4) or not (X31) (Dumonceaux et al., 1999). Fusion efficiency was analyzed by Luciferase activity of cell lysates at different times upon doxycycline
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induction. Results from two representative rtTa– HeLa clones expressing either wtNDK (H-wtNDK) or m7NDK (H-m7NDK) en6 genes are shown in Fig. 1. With the CD4-positive indicator X31CD4 cell line (panel A), the Luciferase activity was clearly detected from 24 h after addition of doxycycline and then increased to reach an almost constant level after 72 h. Activities increased up to 40 fold for H-wtNDK cells and up to 35 fold for H-m7NDK ones. With the CD4negative indicator X31 cell line (panel B), Luciferase activity was only detected after coculture with H-m7NDK cells and followed a similar timecourse, up to a 9-fold increase. As previously described, even if the m7NDK gp120 exhibits a CD4-independent tropism, cell fusion is more effi-
Fig. 1. Kinetic analysis of Env expression by coculture experiments. CD4-dependent (H-wtNDK) or CD4-independent (Hm7NDK) Env expression cell lines were induced by addition of doxycycline. Cell fusion efficiency was estimated at different times after induction and after cocultures either with X31CD4 (U373-MG, LTR-Luc, CXCR4+, CD4+) (Panel A) or with X31 (U373-MG, LTR-Luc, CXCR4+) (Panel B) followed by Luciferase measurements.
cient in the presence of CD4 (Dumonceaux et al., 1998). It should be noted that during this short time-course expression, the CD4-independent phenotype was clearly maintained, as expected (Fig. 1B). The time-course induction of envelope expression was also analyzed at RNA and protein levels. Northern blot hybridization experiments (Fig. 2A) were carried out with total RNA. Three RNAs were detected, the larger one (3.4 kb) corresponding to the en6 mRNA and smaller species (2.6 and 2.0 kb) corresponding to auxiliary genes mRNAs (Fig. 2A). The en6 mRNA was detectable from 16 h after induction and its amount regularly increased to become maximal after 96 h, which was confirmed after monitoring of related GAPDH mRNA levels (Fig. 2A). Western blot experiments (Fig. 2B), were carried out on cell lysates using the monoclonal anti-gp120 Ab D7324. In our experiments, only the gp120 env protein was detected, which suggests that the epitope does not present the same conformation in the gp160 env precursor. The gp120 protein was slightly detectable 16 h after induction and exhibited a regular increase up to 96 h to reach an almost constant level later on. These different time-course analyses demonstrated clearly that gp120 expression could be controlled by the addition of doxycycline. Long-term induction (up to 3 months) was then carried out and Env expression was regularly assayed for both H-m7NDK and H-wtNDK cells. It should be noted that the level reached after long-term induction was similar to that of constitutively expressed wNDK gp120 (data not shown). During these long-term inductions, cells proliferated normally, autofusion events in the H-m7NDK cultures were observed and the envelope gene was always expressed (Fig. 3). Moreover, the CD4-independent phenotype remained unaltered (Fig. 3).
4. Discussion Inducible HIV Env expressing cell lines, tightly controlled through doxycycline induction were engineered and characterised. Indeed, no basal Env-
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Fig. 2. Time-course analysis of HIV or GAPDH specific RNA expression (A) and Western-Blot analysis of gp120 kinetics of expression (B). (A) Total RNA were isolated either from the H-m7NDK or the H-wtNDK cell lines, at different times after doxycycline induction (lane 1 =0 h; 2 =4 h; 3= 8 h; 4= 16 h; 5 = 24 h; 6 =48 h; 7 = 72 h; 8 =96 h and 9 =1 week). Membranes were hybridized either with an HIV-1 en6, tat, re6, 6pu or GAPDH-specific labeled probe, as indicated. These results are representative of three different experiments. (B) Cell lysates were prepared at different times after doxycycline induction (lane 1 =0 h; 2 =16 h; 3= 24 h; 4 = 48 h; 5=72 h; 6 =96 h; 7 = 1 week) from CD4-dependent (HwtNDK) or CD4-independent (H-m7NDK) Env-expressing cell lines, as indicated. Env inducible expressed gp120 were analyzed after incubation with a monoclonal anti-gp120 (D7324). These results are representative of three different experiments.
dependent fusion could be observed before addition of the inducer (Fig. 1 and not shown). The maximum expression level was obtained within 72–96 h thus suggesting a progressive expression of the en6 gene. Moreover, FACS analysis of gp120 expression using a polyclonal human antiserum did not indicate any difference between both m7gp120 and wtgp120. This expression level remained unchanged after an induction period of 3 months (data not shown). A similar time-course for Env proteins inducible expression has been described previously (Cao et al., 1996). This delayed expression of Env proteins seem to result from post-transcriptional events since in the same rtTA-cell lines it was observed that the doxycycline-inducible luciferase reporter construct (Gossen and Bujard, 1992) reached a maximum expression level as soon as 18 h (data not shown). Nevertheless, the delayed expression of Env proteins could not be correlated with a preferential early expression of auxiliary gene mRNAs, as could have been expected (Cullen, 1993).
The first concern was to assay the effects of concomitant expression of a CD4-independent envelope protein and CXCR4 and to determine if direct gp120/CXCR4 interaction could affect cell survival. During time-course experiments no critical alteration of cell proliferation could be observed. H-wtNDK and H-m7NDK cell cultures were carried out similarly at low density to avoid, as much as possible, autofusion in the H-m7NDK cultures. Moreover, the CD4-independent phenotype remained unaltered (Fig. 3), even though it could have been hypothesized that cell survival might have been associated with an extinction of the expression of either CD4-independent envelope or of CXCR4 genes (Valente et al., 2001). Indeed, FACS analysis of both m7NDK and wtNDK gp120s and CXCR4 expression did not indicate any modification during the whole period (data not shown). However, the results with Env inducible expression cell lines contradict our initial findings that clones which expressed m7NDK Env besides
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References
Fig. 3. Analysis of Env expression by coculture experiments after long-term induction. CD4-dependent (H-wtNDK) or CD4-independent (H-m7NDK) Env inducible expression cell lines were induced by addition of doxycycline either for several days or several months. Cell fusion efficiencies were estimated at different times after induction and after cocultures either with X31CD4 (U373-MG, LTR-Luc, CXCR4+, CD4+) or with X31 (U373-MG, LTR-Luc, CXCR4+) followed by Luciferase measurements. Only two representative time-course results are shown for each of the inducible cell lines.
CXCR4 were not viable. Finally, we described a CD4-independent HIV-1-Env expression system in which transcription is directed from an inducible heterologous promoter allowing alternative splicing. Env inducible expression cell lines could constitute a useful cellular tool for carrying with analysis of HIV Env protein expression.
Acknowledgements We are grateful for the technical support of C. Montigny and L. Bougne`res. N. Boord edited the English. J.D. hold a fellowship from Ministe`re de l’Education Nationale, de l’Enseignement Supe´ neur et de la Recherche. A.R. was supported by a grant from Fondation des Treilles. This work was supported by grants from Agence Nationale de la Recherche contre le SIDA (ANRS), ‘Ensemble Contre le SIDA’ AO11, by Fondation Pour la Recherche Me´ dicale and by ‘Lutte AntiSida’ AO2 of the University of Paris 7-Denis Diderot.
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