INTRODUCTION

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Recombinant adenoviruses are one of the preferred viral vectors used in gene therapy, cancer treatment, and recombinant-protein production. Adenoviruses have several advantages that make them suitable for gene transfer experiments (for reviews, see references 8 and 9). For example, the virus is relatively easy to manipulate in vitro and replicates efficiently in permissive cells, thus enabling easy production of high-titer virus stocks. Also, the cell surface receptor for adenovirus (reviewed in reference 15) is expressed on most cells, making it possible to infect a wide range of cell types.

In many protocols, the gene of interest is reconstructed into transcription units that are under the control of a strong promoter, such as the promiscuous cytomegalovirus (CMV) promoter. This ensures that the transgene is expressed at a high level in many cell types. However, expression of endogenous genes in a cell is typically subject to an intricate regulation in response to different stimuli. Thus, constitutive high-level gene expression of a transgene may not be physiological and may interfere with signaling systems in the cell and lead to cellular toxicity. In fact, it has been technically difficult, or impossible, to reconstruct recombinant adenoviruses carrying genes for cytotoxic products, such as the vesicular stomatitis virus G protein (21), the Fas ligand (17), human tumor necrosis factor alpha (10), and the rabies virus glycoprotein (12), using constitutively active promoters, because a high level of expression of these proteins is toxic for the cell. It is noteworthy that construction of a recombinant virus expressing the Fas ligand from an inducible Tet promoter was shown to yield low-titer virus stocks, because even the basal level of Fas ligand expression in 293 cells resulted in apoptosis of the virus-producing cells (17). To produce this virus, it was necessary to establish a 293 cell line that was resistant to apoptosis. It is likely that the problem of transgene interference with virus growth is a more general problem that most often manifests itself as a difficulty in producing high-titer stocks of recombinant adenoviruses.

To overcome this problem, we previously designed recombinant adenovirus vector systems expressing the transgene from gene cassettes regulated by inducible promoters (14). However, during our recent work (13), in which we characterized the effect of overexpression of the essential serine- and arginine-rich (SR) protein ASF/SF2 on adenovirus replication, we became aware of an unexpected complexity of our adenovirus-mediated inducible systems. Infecting 293 cells with a recombinant virus expressing ASF/SF2 from the progesterone antagonist-induced gene expression system (hereafter referred to as the Prog system) resulted in a troublesome background expression of ASF/SF2 in the absence of inducer late after infection (13). Since ASF/SF2 has negative effects, particularly on adenovirus alternative RNA splicing, this background expression was sufficient to significantly perturb late viral mRNA accumulation. 293 cells (5) express the adenovirus E1A and E1B proteins, and this is the producer cell line most commonly used to amplify E1-deficient adenovirus vectors. Here we show that the background expression of ASF/SF2 in 293 cells most likely results from the E1A-289R protein (reviewed in reference 2) activating transcription from the minimal TATA promoter element used in the Prog system. Since the number of potential templates for transcription increases tremendously due to viral DNA replication late after infection, even a low level of E1A-mediated transcription initiation may cause a significant amount of background transgene expression.

We and others have previously used inducible promoters to control transgene expression (for examples, see references 10, 14, 17, and 20), and in one study, the Lac repressor system was used to reduce transgene expression from the constitutively active CMV promoter (12). Here we combine both regulatory systems into a powerful inducible gene cassette. We show that combining the inducible Prog promoter with a Lac repressor protein-based block to transcription elongation dramatically reduces basal ASF/SF2 expression in a 293 cell line expressing the Lac repressor protein. The effect was observed at the level of ASF/SF2 protein expression as well as in a significant relief of the previously observed phenotypic alterations of ASF/SF2 in late viral mRNA accumulation (13).

Collectively, our results show that combining an inducible promoter with a block to transcription elongation results in a dramatic reduction in background transgene expression during virus growth in the 293 producer cell line. Thus, this may be an important factor that will increase the success rate in construction and production of high-titer stocks of recombinant viruses expressing toxic proteins that for specific or general reasons have a strong negative effect on virus multiplication.

	MATERIALS AND METHODS

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Establishment of a stable 293 cell line expressing LacI. 293 cells were transfected with plasmid pCMVLacI (Stratagene) by using the Lipofectamine reagent (Gibco-BRL). The plasmid contains a hygromycin resistance marker and codes for a LacI protein fused to a nuclear localization signal (3). Selection for stably transfected cells was initiated 72 h posttransfection by the addition of hygromycin B (200 µg/ml) to the growth medium. After 7 days, when all 293 cells in an untransfected control dish were dead, the surviving colonies of 293-LacI cells were transferred to a 96-well plate, where they were extended in hygromycin-free medium. Clones that were shown to express the LacI protein were further characterized by transfection of plasmid pOP13CAT (Stratagene), which contains three Lac operator sequences positioned upstream of the chloramphenicol acetyltransferase (CAT) coding sequence. One cell line (denoted 293-LacI) was selected for further experimentation based on its low level of basal expression of CAT combined with a high level of CAT induced by addition of IPTG (isopropyl--D-thiogalactopyranoside) to the culture medium.

Construction of a double-regulated recombinant adenovirus expressing ASF/SF2. The transfer plasmid pAdG5Trip(His)-ASF(Lac) was constructed by cloning a BglII/NotI fragment taken from pOP13CAT (Stratagene) into the MluI site in the -globin intron in plasmid pAdG5Trip(His)-ASF (13). The BglII/NotI fragment contains three lac operator sequences. Plasmid pAdG5Trip(His)-ASF(Lac) was reconstructed into a recombinant adenovirus essentially as described by Stow (18). Briefly, a vector arm was produced by double digestion of genomic Ad5-dl309 DNA with XbaI and ClaI, followed by sucrose gradient purification of the long right-hand 3.71- to 100-unit genomic fragment. Recombinant viruses were generated by in vivo recombination. Thus, 5 µg of pAdG₅Trip(His)- ASF(Lac) DNA was mixed with 1 µg of Ad5-dl309 vector arm and cotransfected by the calcium phosphate coprecipitation technique to 293 cells (5). Plaques typically appeared 5 to 6 days posttransfection. Recombinant viruses were verified by restriction enzyme cleavage of Hirt-extracted viral DNA (6). One positive plaque was selected and purified by a second-round plaque assay. The final reporter virus was named AdG5(His)-ASF(Lac). The activator virus AdCMVProg has been described previously (14).

Purification of recombinant adenovirus. High-titer stocks of recombinant viruses were produced essentially as described previously (7). Briefly, six 15-cm-diameter plates of 293 cells were infected with approximately 5 PFU of recombinant virus per cell. Three days postinfection (p.i.), when a clear cytopathic effect was visible, cells were harvested by low-speed centrifugation. The cell pellet was freeze-thawed once and resuspended in 2 ml of 0.1 M Tris-HCl, pH 8.0, followed by lysis with 0.1 volume of 5% Na-deoxycholate on ice for 30 min. Subsequently, the cell lysate was sonicated on ice, and virus was purified by CsCl centrifugation. The virus band was collected and dialyzed against 100 volumes of phosphate-buffered saline containing 1 mM CaCl₂, 1 mM MgCl₂, and 10% glycerol, using a Slide-A-Lyzer cassette (Pierce). The virus titer was determined by plaque assay. Typical virus titers were 10¹⁰ PFU/ml.

Infections. For infections, virus stocks were diluted in 1 ml of Dulbecco's modified Eagle's medium supplemented with 2% NCS and used to infect one subconfluent 6-cm-diameter plate of 293 or 293-LacI cells, except for one experiment (see Fig. 3) in which a U2OS cell line expressing the adenovirus E1A transcription unit under the transcriptional control of a Tet-ON-regulated promoter (see reference 14; kindly provided by C. Svensson) was used. In all experiments, 5 PFU of AdCMVProg and AdG5(His)-ASF or AdG5(His)-ASF(Lac) per cell was used. The inoculum was removed after a 1-h incubation at 37°C, and the cells were washed two times with fresh medium followed by the addition of 4 ml of Dulbecco's modified Eagle's medium supplemented with 10% NCS with or without 0.5 µM RU 486 and/or 5 mM IPTG. Expression of the E1A proteins was induced in the U2OS Tet-E1A cell line (see Fig. 3) by addition of doxycycline to the culture medium to a final concentration of 4 µM 8 h prior to infection.

Western blot analysis. Protein extracts were prepared 18 h p.i. by lysis of infected cells with RIPA buffer (10 mM Tris buffer, pH 7.4, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1× Complete Protease Inhibitor Cocktail [Boehringer-Mannheim]). Extracts were subjected to SDS-12% polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane by electroblotting. His-tagged ASF/SF2 was detected using a six-His monoclonal antibody (Clontech). Filters were treated as previously described (16), and proteins were visualized by chemiluminscence (SuperSignal West Pico; Pierce) as described in the manufacturer's protocol, using a horseradish peroxidase-conjugated secondary antibody.

Northern blot analysis. Total cytoplasmic RNA was prepared by lysis with IsoB-NP-40 (10 mM Tris-HCl [pH 7.9], 150 mM NaCl, 1.5 mM MgCl2, 1% NP-40) followed by two rounds of phenol-chloroform-isoamyl alcohol and one extraction with chloroform-isoamyl alcohol (1). Two micrograms of cytoplasmic RNA was separated on a 1% agarose gel containing 2.2 M formaldehyde, transferred to a nitrocellulose filter, and hybridized with a DNA probe ³²P labeled by random priming as previously described (1). The Ad2 HindIII I fragment (corresponding to adenovirus 31.5 to 37.3 map units) was used as a probe to detect L1 mRNAs.

	RESULTS

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Construction of a novel gene cassette combining an inducible promoter and a Lac repressor protein-based inhibition of transcription elongation. To silence the 293 cell-specific leaky expression of the ASF/SF2 reporter gene from our RU 486-inducible Prog system (13, 14), we introduced the LacSwitch II Inducible Mammalian Expression System (Stratagene) into our viral vector system. The approach relies on the construction of a stable 293 cell line expressing the Lac repressor protein (293-LacI) and construction of a new ASF/SF2 reporter virus in which three Lac operator sequences were inserted into the intron located downstream of the ASF/SF2 gene (Fig. 1). This virus was named AdG5(His)-ASF(Lac). The idea was to use the Lac repressor protein to restrict background ASF/SF2 expression, in the absence of inducer, by preventing production of full-length mRNA from promiscuously initiated transcripts.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Experimental strategy to silence basal transgene expression. The promiscuous E1A transcriptional activator proteins are a major cause of basal transgene expression in 293 cells (top). To block E1A-mediated activation of the basal Prog promoter, three tandem copies of the lac operator (3×LacO) were inserted into the -globin intron located downstream of the ASF/SF2 coding sequence (bottom). The Lac repressor protein expressed in the 293-LacI cell line will reduce basal transgene expression by binding to the LacO sites and prevent synthesis of full-length transcripts in the absence of inducer. ss, splice site; p(A), poly(A).

High-level expression and tight control of ASF/SF2 expression in 293-LacI cells. To determine whether our experimental approach worked as predicted, we compared the level of background ASF/SF2 expression in 293-LacI cells double infected with equal numbers of PFU of an activator and a reporter virus. Thus, activator virus, AdCMVProg (14) encoding a chimeric Gal4-VP16-progesterone transactivator protein, was mixed with reporter virus, AdG5(His)-ASF or AdG5(His)-ASF(Lac) expressing His-ASF/SF2 from a Gal4-regulated promoter. Transcription of the His-ASF/SF2 gene was induced by addition of RU 486 and/or IPTG to the culture medium. IPTG, which has no negative effects on the growth of eukaryotic cells at concentrations below 50 mM (4, 19), decreases the binding affinity of the Lac repressor protein to the Lac operator sequence, thus relieving the inhibitory effect of the Lac repressor protein on transcriptional elongation.

View larger version (43K): [in this window] [in a new window]   FIG. 2.   The 293-LacI cell line markedly reduces uninduced transgene expression. 293-LacI or 293 cells were coinfected with AdCMVProg and AdG5(His)-ASF(Lac) or AdG5(His)-ASF. His-ASF/SF2 expression was induced at the start of infection by the addition of RU 486 to the culture medium. Whole-cell extracts were prepared at 18 h p.i., separated on an SDS-12% PAGE gel, and probed with an anti-His monoclonal antibody to detect His-ASF/SF2 expression. The lower gels show a longer exposure of the filter to make it possible to compare the background expression of His-ASF/SF2 in the absence of inducer in 293-LacI and 293 cells. + denotes addition of virus and/or inducer.

The adenovirus E1A proteins activate basal transgene expression. The E1A transcriptional activator proteins expressed in the 293 cell line have previously been shown to promiscuously activate transcription from basal promoter elements (reviewed in reference 2). We predicted that E1A-mediated activation of the basal Prog promoter in the parental AdG5(His)-ASF virus was responsible for the low level of basal expression of His-ASF/SF2 that we previously observed in the 293 cell line (13) (Fig. 2). To test this hypothesis, we infected a U2OS cell line expressing the adenovirus E1A proteins under the inducible control of a Tet-ON-regulated promoter with the AdG5(His)-ASF virus.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Effect of adenovirus E1A on uninduced transgene expression. AdG5(His)-ASF with or without AdCMVProg was used to infect a U2OS cell line expressing the adenovirus E1A transcription unit from a Tet-ON-inducible promoter. E1A expression was induced 8 h prior to infection by addition of doxycycline (Dox) to the culture medium. Whole-cell extracts were prepared 18 h p.i. Fifty micrograms of total protein was separated on an SDS-12% PAGE gel and probed with an anti-His monoclonal antibody. Lanes 1 and 4, mock-infected cells. + denotes addition of virus and/or inducer.

ASF/SF2 inhibition of adenovirus alternative RNA splicing is to a large extent relieved in 293-LacI cells. We previously showed that overexpression of the prototypical SR protein ASF/SF2 during lytic growth of adenovirus almost completely blocked expression of mRNAs from the major late transcription unit (13). In addition, we showed that the basal (uninduced) expression of His-ASF/SF2 in AdG5(His)-ASF-infected cells was sufficient to almost completely inhibit late specific IIIa mRNA expression in 293 cells (13). As shown in Fig. 2, infection of 293-LacI cells with the modified AdG5(His)-ASF(Lac) virus strongly attenuates basal ASF/SF2 expression. This reduction in basal ASF/SF2 expression would be predicted to also relieve the negative effects of background ASF/SF2 expression on L1 alternative splicing. To test this hypothesis, we compared the L1 mRNA profile in AdG5(His)-ASF- and AdG5(His)-ASF(Lac)-infected 293-LacI cells.

View larger version (46K): [in this window] [in a new window]   FIG. 4.   Northern blot analysis of the adenovirus major late region 1 mRNA expression. (A) Schematic representation of the alternatively spliced mRNAs produced from the L1 transcription unit. L1 pre-mRNA splicing is temporally regulated, with different mRNA species accumulating at early and late times of infection (reviewed in reference 11). (B) 293-LacI cells were coinfected with AdCMVProg and AdG5(His)-ASF(Lac) or AdG5(His)-ASF. Cytoplasmic RNA was prepared 18 h p.i. Two micrograms of total cytoplasmic RNA was separated on a denaturing 1% agarose gel and subsequently transferred to a nitrocellulose filter. 52,55K and IIIa mRNA expression was detected by hybridization with an L1-specific 32P-labeled probe followed by autoradiography. Lane 1, mock-infected cells. + denotes addition of virus and/or inducer.

The LacI repressor protein does not interfere with transgene expression from a recombinant virus grown in 293-LacI cells. The Lac repressor protein is a DNA binding protein and as such might be incorporated into the AdG5(His)-ASF(Lac) virus capsid during virus propagation in 293-LacI cells. This is an important issue, since Lac repressor protein binding to viral DNA encapsidated in new virus particles might restrict transgene expression and therefore require inclusion of IPTG in subsequent gene transfer protocols. To check whether this was necessary, an AdG5(His)-ASF(Lac) virus stock grown in 293-LacI cells was used to infect HeLa cells. As shown in Fig. 5, addition of RU 486 resulted in a strong activation of His-ASF/SF2 expression, an activation that was not further increased by addition of IPTG (compare lanes 3 and 4). This result suggests that the LacI repressor protein does not become incorporated to a significant degree into mature virus particles as a protein binding to the Lac operator sequences.

View larger version (24K): [in this window] [in a new window]   FIG. 5.   The Lac repressor protein does not interfere with transgene expression from a virus grown in 293-LacI cells. HeLa cells were coinfected with an AdG5(His)-ASF(Lac) virus stock produced in 293-LacI cells and AdCMVProg in the presence or absence of IPTG. Whole-cell lysates were prepared at 18 h p.i. One hundred micrograms of total protein was separated on an SDS-12% PAGE gel and probed with an anti-His monoclonal antibody detecting ASF/SF2. + denotes addition of virus and/or inducer.

	DISCUSSION

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We have previously shown that the RU 486-inducible Prog vector system functions extraordinarily well in cell lines where the virus does not replicate, reaching induction levels exceeding 600-fold (14). However, during our work with a recombinant virus expressing the essential splicing factor ASF/SF2 (13), we noted that the transgene was significantly expressed in the 293 cell line, even in the absence of inducer, causing significant abnormalities in late gene expression (13). Since 293 is the standard cell line used for propagation of recombinant viruses, this background expression of the transgene may cause problems and reduce the virus yield or, in the worst case, prevent reconstruction of viruses expressing toxic proteins (see the introduction).

Here we show that this troublesome background of transgene expression is caused by the E1A transcriptional activator proteins expressed in 293 cells, which most likely activate transcription from the TATA element present in the G5 minimal major late promoter driving reporter gene expression (Fig. 3). Since viral DNA replication results in a tremendous increase in the number of DNA templates available for transcription, even a low level of E1A-mediated activation of transgene expression may become troublesome at late times during infection. We reasoned that it would be impossible to completely block the activity of the promiscuous E1A transcriptional activator proteins in the 293 cell line by use of alternative minimal promoter elements, since the E1A-289R protein has been shown to activate transcription from a basal TATA promoter (reviewed in reference 2). We therefore argued that combining two inducible systems, based on different principles, might be a better way to tighten the control of gene expression. Here we show that combining a LacI-based block to transcription elongation and the inducible Prog-regulated gene cassette indeed resulted in a dramatic reduction in the background expression of the transgene in the 293-LacI cell line (Fig. 2). This reduction in leaky transgene expression should increase the success rate when recombinant adenoviruses expressing proteins that are highly toxic to virus multiplication are constructed (see the introduction). Also, this system has the advantage that it should allow for production of high-titer stocks of recombinant viruses, which express proteins that, for specific or general reasons, interfere with virus multiplication.

Admittedly, ASF/SF2 is not the ideal reporter gene in this set of experiments, since ASF/SF2 overexpression is only moderately toxic to adenovirus replication (13), causing a 60-fold reduction in virus yield at maximum induction. Also, growth of the AdG5(His)-ASF(Lac) virus in 293-LacI cells caused a modest twofold increase in virus yield compared to normal 293 cells (data not shown). Nevertheless, we believe that our work using the ASF/SF virus proves the principle that combining an inducible promoter and a block to transcription elongation is an effective technique to tighten the control of transgene expression. However, in its present version, this system does not result in a complete shutoff of background transgene expression (Fig. 2, lane 4, longer exposure, and Fig. 4B, lane 4). Our current experiments are aimed at refining the expression system to further suppress background transgene expression.