Generation of a Flexible Cell Line with Regulatable, High-Level Expression of HIV Gag/Pol Particles Capable of Packaging HIV-Derived Vectors  Sandra Sparacio, Tanya Pfeiffer, Heiner Schaal, Valerie Bosch  Molecular Therapy  Volume 3, Issue 4, Pages 602-612 (April 2001) DOI: 10.1006/mthe.2001.0296 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Expression scheme indicating the vectors (described under Materials and Methods) employed for the generation of stable cell lines inducibly expressing HIV Gag/Pol particles for the packaging of HIV-derived vectors. Promoters, as indicated, are depicted as empty bars; coding sequences, as indicated, as gray bars; and essential cis elements (IRES, RRE) as black bars. In the vector pIND-Rev-IRES-Rev (not depicted), the Tat sequence has been replaced with a further Rev sequence. Molecular Therapy 2001 3, 602-612DOI: (10.1006/mthe.2001.0296) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Inducible expression of the HIV regulatory proteins Rev and Tat in the stable cell lines 293-Tat/Revi and 293-Revi. (A) 293 cells (empty columns) were transfected with pNL4-3 (lane 1) or the tat- and rev-defective proviral construct pNL4-3tat−/rev− (lane 2), 293-Tat/Revi cells (hatched columns) or 293-Revi cells (gray columns) were transfected with pNL4-3tat−/rev− (plus pCMVtat in the case of 293-Revi cells) and cultured for 48 h in the absence (–) (columns 3 and 5) or presence (+) (columns 4 and 6) of 1 iM ponasterone A. (B) 293-Tat/Revi cells (hatched columns) or 293-Revi cells (gray columns) were transfected with pIND(Sp1)-Δψgag/pol-RRE and cultivated in the absence (–) or presence (+) of 1 iM ponasterone A. HIV Gag/Pol particles release into the culture supernatant were quantitated by ELISA for CA (given as pg/ml p24). Note that, due to relatively low transient transfection efficiencies, the maximal amounts of CA expressed here (in the range of 0.2 ig/ml) are lower than in subsequent analyses. Molecular Therapy 2001 3, 602-612DOI: (10.1006/mthe.2001.0296) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Inducible HIV Gag/Pol particle expression in a cell line derived by stable transfection of 293-Tat/Revi cells with pIND(Sp1)-Δψgag/pol-RRE in the absence of Saquinavir. (A) Indirect immunofluorescence, employing antibodies against HIV CA, of cells after cultivation for 48 h in the absence (–) or presence (+) of 1 μM ponasterone A as indicated. (B) Amounts (given in pg/ml) of HIV Gag/Pol particles in the media of uninduced (–) and induced (+) cells quantitated by ELISA for CA. (C) Western blot analyses, employing antibodies against HIV p24, of cell lysates and of pelleted particles from media of uninduced (–) and induced (+) cells. The positions of the HIV Gag proteins are indicated Molecular Therapy 2001 3, 602-612DOI: (10.1006/mthe.2001.0296) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Inducible HIV Gag/Pol particle expression in 293-Rev/Gag/Pot cells. (A) Indirect immunofluorescence, employing antibodies against HIV CA, of cells after cultivation for 48 h in the absence (–) or presence (+) of 1 xM ponasterone A as indicated. (B) Gel electrophoresis and autoradiography (no immunoprecipitation) of metabolically labeled HIV Gag/Pol particles centrifuged from culture supernatants of uninduced (–) and induced (+) 293-Rev/Gag/Poli cells. The positions of HIV Gag/Pol proteins are shown on the left and of molecular weight markers on the right. Molecular Therapy 2001 3, 602-612DOI: (10.1006/mthe.2001.0296) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 HIV Gag/Pol particle production and cell growth of uninduced and induced 293-Rev/Gag/Poli cells having been maintained in culture for different lengths of time. Cultures consisting of 2 × 105 293-Rev/Gag/Poli cells either shortly after thawing (1 week) (filled-in columns) or after maintenance in culture for 6 weeks subsequent to thawing (open columns) were plated in the presence (+/) or absence (-/) of 2 ponasterone A. 24 h later, the indicated cultures were (/+) or were not (/-) treated for 18 h with 5 mM sodium butyrate as indicated. Subsequent to the sodium butyrate treatment, the media in all cultures were replaced with fresh medium (with or without ponasterone A). 48 h subsequent to this, the amounts of HIV Gag/Pol particles in the media were quantitated by ELISA for CA, the numbers of cells in the respective cultures were counted, and the processing status of the expressed HIV Gag proteins in the cell lysates and culture supernatants was analyzed by Western blot. (A) Amounts of p24 in the media of the indicated cultures in ng/ml (amounts noted above each column). (B) Cell number in the respective cultures (cell number × 104 noted above each column). The horizontal line indicates the cell density (2 × 105 cells) at plating. (C) Western blot analyses, employing antibodies against HIV p24, of cell lysates (left) and of culture supernatants (right) of cultures 1, 2, and 3. The positions of the HIV Gag proteins are indicated. Molecular Therapy 2001 3, 602-612DOI: (10.1006/mthe.2001.0296) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 Effects of concentration of ponasterone A and induction time on the amounts of HIV Gag/Pol particles released from 293-Rev/Gag/Poli cells. (A) 293-Rev/Gag/Poli cells were induced with the indicated concentrations of ponasterone A in the absence (⋄) or presence (▪) of additional treatment (for the first 18 h of induction) with 5 mM sodium butyrate. The amounts of p24 in the culture supernatants were quantitated by ELISA either 2 days postinduction (⋄) or 2 days after treatment with sodium butyrate (▪). (B) 293-Rev/Gag/Poli cells were induced with 1 xM ponasterone A (open columns) or were induced with 2 xM ponasterone A and simultaneously treated with 5 mM sodium butyrate for 18 h (filled-in columns). In the latter case, the medium was then replaced with fresh medium containing 2 xM ponasterone A but lacking sodium butyrate. Subsequent to this, aliquots of the culture supernatant were collected at daily intervals and the amounts of HIV Gag/Pol particles released quantitated by ELISA for CA. Molecular Therapy 2001 3, 602-612DOI: (10.1006/mthe.2001.0296) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 7 FACS analysis of HeLa cells transduced with GFP-expressing vector, pRRL-eGFP (7), packaged by HIV Gag/Pol particles and VSV-G produced from induced 293-Rev/Gag/Poli cells. 293-Rev/Gag/Poli cells were transiently transfected with pRRL-eGFP (7) with (A, B) or without (C) pMD.G (19) and 4 h later, the cultures were (A) or were not (B, C) treated with 2 μM ponasterone A and 5 mM sodium butyrate for 16 h. Subsequently the media were replaced with fresh medium lacking sodium butyrate but containing (A) or not containing (B, C) 2 xM ponasterone A. 24 h later the media were applied to a defined number of HeLa cells in the presence of 8 xg/ml Polybrene. 48 h later, untransduced and transduced HeLa cells were analyzed for GFP expression by FACS. In the control cells (empty peak, untransduced HeLa cells), 0.19% of the cells exhibit fluorescence within the indicated range. The black regions indicate the fluorescence profiles obtained on transduction of 1.3 × 105 HeLa cells with 1 ml medium from the respective 293-Rev/Gag/Poli cell cultures. Molecular Therapy 2001 3, 602-612DOI: (10.1006/mthe.2001.0296) Copyright © 2001 American Society for Gene Therapy Terms and Conditions