JOURNAL OF VIROLOGY, Nov. 2001, p. 10041–10053 0022-538X/01/$04.00⫹0 DOI: 10.1128/JVI.75.21.10041–10053.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 21
Overexpression of the Adenovirus Type 12 (Ad12) pTP or E1A Gene Facilitates Ad12 DNA Replication in Nonpermissive BHK21 Hamster Cells ¨ SEL, DENNIS WEBB, JO ¨ RG SCHRO ¨ ER,† BIRGIT SCHMITZ, MARIANNA HO AND WALTER DOERFLER* Institut fu ¨r Genetik, Universita ¨t zu Ko ¨ln, D-50931 Cologne, Germany Received 20 February 2001/Accepted 7 August 2001
In the adenovirus type 12 (Ad12) hamster cell system, abortive virus infection is one of the factors associated with the highly efficient oncogenesis in newborn Syrian hamsters. We have shown earlier that the replication and efficient late transcription of the Ad12 genome are blocked in Syrian hamster cells. Some of the early Ad12 functions are transcribed in these cells, although at a minimal rate. In the present study, we demonstrate that low expression levels of the E1A and precursor to terminal protein (pTP) genes of Ad12 seem to be responsible for the lack of Ad12 DNA replication in hamster cells. The Ad12 genes for the E1A functions or for pTP were tethered to the strong early promoter of the human cytomegalovirus and transfected into BHK21 cells. Subsequently, these cells were infected with Ad12 virions. In Ad12-infected BHK21 cells, which overexpressed pTP or E1A, full-length Ad12 DNA was de novo synthesized, as documented by metabolic labeling with [3H]thymidine and by zone velocity sedimentation in alkaline sucrose gradients followed by gel electrophoresis of the 3H-labeled DNA and Southern blot hybridization to 32P-labeled Ad12 DNA. Transfection of the cloned E1A region of Ad2 yielded similar results. The newly synthesized Ad12 DNA was covalently linked to pTP. The Ad12 DNA binding protein (DBP) and DNA polymerase (pol) genes were transcribed at levels similar to those in merely Ad12-infected cells. In pTP or E1A gene-transfected and Ad12-infected BHK21 cells, marginal levels of late Ad12 mRNA were detectable. Late Ad12 proteins were, however, not synthesized. Apparently, Ad12 DNA replication in hamster cells is rendered impossible due to insufficient threshold levels of the viral E1A and/or pTP. The array of cellular and viral genetic functions expressed in a cell preceding, during, and subsequent to viral infection is decisive for the outcome of individual virus-cell interactions. Syrian hamster cells, such as the BHK21 cell line or primary Syrian hamster cells, are permissive for the replication of human adenovirus type 2 (Ad2) at moderate levels (30). In contrast, the replication of human Ad12 is totally blocked in BHK21 Syrian hamster cells (6, 29). Studies of the interaction of Ad12 with Syrian hamsters are of particular interest because of the high oncogenic potential of this virus in newborn Syrian hamsters (10, 17, 31). In BHK21 cells, Ad12 DNA replication cannot proceed, and late viral functions are not detectably transcribed, whereas some of the early Ad12 genes are expressed, many at reduced rates (for reviews, see references 6, 11, 20, 25, and 35). Ad12 precursor to terminal protein (pTP) and the Ad12 DNA polymerase can associate in extracts from Ad12-infected BHK21 cells in vitro to form an initiation complex of Ad12 DNA, although chain elongation never proceeds. Moreover, this initiation activity is markedly lower in BHK21 cell extracts than that achieved with extracts from Ad12 productively infected human KB cells (5, 20). The data now available on this abortive system suggest there are stops or inefficiencies at several levels in the viral replication cycle. In addition, there is a negative regulatory or mitigator element
downstream of the major late promoter region of Ad12 DNA (34). The E1 functions of Ad5, the genes of which are integrated and constitutively expressed in the Ad5-transformed hamster cell line BHK297-C131, can help to partly overcome the blocks in Ad12 DNA replication and late transcription (14, 15), but not in late mRNA translation (25). We have now demonstrated that the pTP and E1A functions of Ad12 are minimally expressed in abortively infected BHK21 cells. We have, therefore, investigated the effects of transfecting and overexpressing the early Ad12 function E1A or pTP in BHK21 cells that have subsequently been infected with Ad12. E1A is the paradigm viral transactivator of all viral (2, 13, 21, 26) and numerous cellular promoters. pTP, the precursor for the Ad12 terminal protein (TP), an E2B function, plays an important role in the initiation of adenovirus DNA replication (22; for review, see reference 32) and in the interaction of the viral DNA with the nuclear matrix (9, 24). BHK21 cells transfected with the pTP or the E1A gene of Ad12 or the E1A gene of Ad2 are capable of synthesizing moderate amounts of fulllength Ad12 DNA. The detection by reverse transcriptionPCR (RT-PCR) of late Ad12 transcripts in pTP or E1A genetransfected and Ad12-infected BHK21 cells is marginal. Late Ad12 proteins are not made in this substituted system.
* Corresponding author. Mailing address: Institut fu ¨r Genetik, Universita¨t zu Ko ¨ln, Weyertal 121, D-50931 Cologne, Germany. Phone: 49-221-470-2386. Fax: 49-221-470-5163. E-mail:
[email protected] .uni-koeln.de. † Present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014.
Cell lines, virus, and virus infection. Human HeLa cells, the baby hamster kidney cell line BHK21, or the Ad5-transformed hamster cell line BHK297-C131 (C131) (33) were grown on monolayer cultures in Dulbecco medium (1) supplemented with 10% fetal calf serum. Human Ad2 and Ad12 were propagated in HeLa cells and purified as described previously (6). For the infection with Ad2 or Ad12, cells were grown on monolayers to 40 to 50% confluence. HeLa cells
MATERIALS AND METHODS
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were infected with 20 to 25 PFU of CsCl-purified Ad12 per cell. C131 cells were inoculated with 75 to 100 PFU of Ad12. BHK21 cells were infected with 20 to 25 or with 75 to 100 PFU of Ad2 or Ad12 per cell, respectively. Construction of the expression vectors and transfection of BHK21 cells. The 871-bp fragment corresponding to the E1A gene of Ad12, the 984-bp fragment corresponding to the E1A gene of Ad2, or the 1,821-bp fragment corresponding to the pTP gene of Ad12 was amplified by PCR (23) with the 12E1Af and 12E1Ar, 2E1Af and 2E1Ar, or the pTPf and pTPr primers, respectively (described below). These primers contained the following sequences for the NheI and BamHI restriction sites (indicated by underlining), respectively: 12E1Af, 5⬘-GAAGCTAGCATGAGAACTGAAATGACTCCC-3⬘ (f ⫽ forward); 12E1Ar, 5⬘-GACGGATCCTTACATCTAGGGCGTTTCAC-3⬘ (r ⫽ reverse); 2E1Af, 5⬘GAAGCTAGCATGAGACATATTATCTGCCAC-3⬘; 2E1Ar, 5⬘-GACGGATC CTTATGGCCTGGGGCGTTTAC-3⬘; pTPf, 5⬘-GAAGCTAGCATGCGAGC AACAACTACC-3⬘; and pTPr, 5⬘-AAAGGATCCTTAAAATCGGCGGCGCG GAC-5⬘. The PCR fragments thus generated were cloned into the NheI and BamHI sites of the expression vector pEGFP-C1 (Clontech). In this vector, the enhanced green fluorescent protein (EGFP) gene was under the control of the human cytomegalovirus (HCMV) promoter. Previously, the pEGFP-C1 vector had been cut with NheI and BamHI to excise the EGFP gene and to prepare the vector for the insertion of PCR products. For electroporation, BHK21 cells were grown to about half-confluence on monolayer cultures. About 2.5 ⫻ 106 cells were suspended in 0.5 ml of RPMI medium without glutamine or dye (Life Technologies, Inc.). Ten micrograms of the different plasmid constructs plus 3 g of the pEGFP-C1 vector were added, and the mixture was electroporated in 4-mmdiameter cuvettes with a Bio-Rad gene pulser at 300 V, 960 F at room temperature. The cells were then incubated at 37°C in 60-mm-diameter dishes in Dulbecco medium supplemented with 10% fetal calf serum. At 18 h after electroporation, the BHK21 cells were washed twice in phosphate-buffered saline without Mg2⫹ and Ca2⫹ (PBSd) (8) and mock infected or Ad12 infected as described above. Subsequently, DNA, RNA, or protein was extracted from these cells at different times postinfection (p.i.). Southern blot analyses. At different times p.i., the total cellular DNA was extracted from BHK21 cells, which were mock or Ad12 infected or which had previously been transfected with different constructs and then subsequently infected with Ad12. For DNA preparation, a Qiagen genomic purification kit was used. Ten-microgram amounts of DNA were cleaved with EcoRI, and the fragments were electrophoresed on a 0.7% agarose gel. Upon Southern blot transfer (16, 28) to nylon membranes, Ad12-specific DNA was visualized by hybridization to the 32P-labeled EcoRI-E and EcoRI-F fragments of Ad12 DNA (map in Fig. 5) that had been excised and gel purified from the pBR322 vectors. Analyses of newly synthesized Ad12 DNA in E1A- or pTP-transfected and Ad12-infected BHK21 cells. At 18 h after electroporation, E1A- or pTP-transfected BHK21 cells were mock or Ad12 infected as described above. Mockinfected BHK21 cells, nontransfected but Ad12-infected BHK21 cells, and mockand Ad12-infected C131 or HeLa cells were investigated as controls. The newly synthesized DNA in these systems was labeled by adding [3H]thymidine (370 GBq/mmol) at a concentration of 35 Ci per ml of medium at 6 h p.i. At 28 h p.i. or after mock infection, about 105 hamster cells or about 2 ⫻ 104 HeLa cells were lysed by adding them to a 0.4-ml top layer of 0.5 N NaOH–10 mM EDTA on a 5 to 20% sucrose gradient in 0.7 M NaCl–0.3 M NaOH–10 mM EDTA in an SW41 rotor tube of a Beckman ultracentrifuge. After completion of lysis at 4°C for 18 h, the samples were centrifuged for 3 h at 35,000 rpm at 4°C. The bottom of the tube was then punctured, and 300-l fractions were collected. The 3H activity in each fraction was determined in a Beckman liquid scintillation counter. The entire procedure was described in detail earlier (4). To assess the nature of the 3H-labeled DNA, portions of the 3H-labeled peak or of the neighboring fractions devoid of 3H label were neutralized with acetic acid, ethanol precipitated, and analyzed by electrophoresis on a 0.7% agarose gel followed by Southern blotting. 32P-labeled Ad12 DNA was used as a hybridization probe. Production of anti-Ad12 pTP serum. The 1,821-bp-long pTP gene of Ad12 was amplified by PCR with TP1 and TP3 primers containing sequences for the BamHI and SmaI restriction sites (indicated by underlining): TP1(5⬘-CCGGAT CCCTATGCGAGCAACAACTACCGCTGCC-3⬘) and TP3 ( 5⬘-GAGCCCGG GTTAAAATCGGCGGCGCGGACGAGCTCC-3⬘). The thus-generated PCR fragment was cloned into the pGEX-3X expression vector (Pharmacia Biotech), and the cloned pTP was expressed in E. coli strain BL21 as a glutathione S-transferase (GST) fusion protein (27). For optimal expression of the GST-pTP protein, overnight cultures were diluted 1:10 in Luria-Bertani (LB) medium in the presence of 100 g of ampicillin per ml. After 1 h at 37°C, the bacterial culture was adjusted to 5 mM IPTG (isopropyl--D-thiogalactopyranoside), fol-
J. VIROL. lowed by 2.5 h of incubation at 37°C. The bacteria were then harvested by centrifugation and resuspended in 50 mM Tris-HCl (pH 8.0). After ultrasonic treatment of the bacterial suspension, cellular proteins were fractionated by electrophoresis on a sodium dodecyl sulfate (SDS)–10% polyacrylamide gel and stained with Coomassie brilliant blue R250. The 93-kDa band corresponding to the GST-pTP fusion protein was excised from the gel and was used for the production of the polyclonal rabbit antibody by Eurogentech (Seraing, Belgium). Western blot analysis. Cells were either mock infected or infected with Ad12 or Ad2, harvested at different times p.i., and solubilized in the T-PER protein extraction reagent (Pierce). After pelleting the cell debris by centrifugation at 10,000 rpm for 5 min, the supernatant was collected, and the protein concentration was determined by the Bradford method (3). Appropriate amounts of protein extracts were fractionated by electrophoresis in SDS-polyacrylamide gels (18) and transferred to Hybond-P polyvinylidene difluoride membranes (Western blotting). Specific proteins were identified by using different primary antibodies. The Ad12 E1A protein was detected with a mouse monoclonal antibody kindly provided by R. Grand, Birmingham, United Kingdom. An Ad2 E1A (13S-5) polyclonal rabbit antibody (Santa Cruz Biotechnology) was used to recognize the Ad2 or Ad5 E1A antigens. The Ad12 fiber protein was detected with the Ad12 fiber rabbit antiserum kindly provided by P. Freimuth, Brookhaven National Laboratory, Upton, N.Y. A GFP (FL) polyclonal rabbit antibody (Santa Cruz Biotechnology) was used to detect the GFP. After the incubation with primary antibodies in Tris-buffered saline (TBS) containing 5% (wt/vol) milk powder and 0.05% (vol/vol) Tween 20, the membranes were washed and incubated with the horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Amersham) and developed with the ECL-plus enhanced chemiluminescence detection system (Amersham). Prior to reprobing, membranes were incubated for 30 min at 60°C in stripping buffer (100 mM -mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl [pH 6.7]) and washed twice in TBS containing 0.05% (vol/vol) Tween 20. Northern blot analysis and RT-PCR. Total RNA from mock- or Ad12-infected cells was purified by using the RNeasy Midi extraction kit (Qiagen). For RNA transfer (Northern blotting) experiments, 20 g of RNA was fractionated by electrophoresis on 1% agarose gels containing 2.2 M formaldehyde. Subsequently, the RNA was transferred to nylon membranes by standard protocols (19). The RNA was then hybridized to a 32P-labeled PCR fragment of Ad12 DNA, which carried the E1A gene of Ad12. For RT-PCR, total RNA was isolated from BHK21 cells that had been transfected with the E1A gene of Ad12, with the E1A gene of Ad2, or with the pTP gene of Ad12 and then infected with Ad12. To analyze for the presence of the Ad12 pTP transcripts, 100 ng of total RNA was subjected to quantitative RTPCR by using primers for the pTP gene 5⬘-AAGTGACGTGTGGGGAATG G-3⬘ (sense) and 5⬘-GAGCCCGGGAACTCCACCTCTAAGTTC-3⬘ (antisense). RNAs from mock- or pEGFP-C1-transfected and Ad12-infected BHK21 cells as well as from mock- or merely Ad12-infected HeLa cells were used as controls. RT-PCR was performed with avian myeloblastosis virus (AMV) reverse transcriptase and Tfl DNA polymerase in the same reaction (Access RT-PCR; Promega). As an internal control, the transcription of the -actin gene was analyzed by coamplification with a second, -actin-specific primer pair: 5⬘-ATG GATGATGATATCGCCGC-3⬘ (sense) and 5⬘-GTGTGGTGCCAGATTTTCT CC-3⬘ (antisense). RT-PCR was carried out with 100 ng of each primer, 1 mM Mg2⫹, a 0.2 mM concentration of each of the four deoxynucleoside triphosphates (dNTPs) in the presence of 0.1 Ci of [␣-32P]dCTP (3,000 Ci/mmol), and 2.5 U of both enzymes in AMV/Tfl buffer (Promega) in a volume of 25 l by using a DNA Thermo Cycler (Perkin-Elmer Cetus). Total RNA from mock-transfected and Ad12-infected BHK21 cells was analyzed in different RT-PCR experiments with 25 to 40 PCR cycles. The synthesis of pTP-specific RT-PCR products in BHK21 cells, which had been abortively infected with Ad12, was first detected after 35 PCR cycles. Temperature cycling was as follows: 48°C for 45 min (RT); 35 cycles at 94°C for 30 s, 63°C for 1 min, and 68°C for 45 s; and 68°C for 5 min, allowing sequential RT and PCR without interruptions. Portions of 6 l of the final 30-l PCR volume were analyzed by electrophoresis on a 4% polyacrylamide gel followed by phosphorimager quantitation of the pTP or -actin RT-PCR products. The intensity values for pTP or -actin were corrected for the background as determined with RNA from mockinfected BHK21 cells that lacked the RT-PCR product of the Ad12 pTP gene. Finally, all values were normalized to the internal -actin standard. BHK21 cells transfected with the pEGFP-C1, with the Ad12-pTP, with the Ad12-E1A, or with the Ad2-E1A construct and subsequently infected with Ad12 were also analyzed for the expression of mRNAs from the Ad12 DBP gene or the Ad12 DNA polymerase gene by RT-PCR. Total RNA was prepared from these cells at 24 or 30 h p.i. RNA isolated from mock- or Ad12-infected BHK21, C131, or HeLa cells was used as a control. RNA was subjected to quantitative RT-PCR
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FIG. 1. Synthesis of pTP and TP in Ad12-infected BHK21, C131, or HeLa cells. Protein extracts from mock-infected (lane 1) or Ad12-infected (lane 2) BHK21 cells or from mock-infected (lane 3) or Ad12-infected (lane 4) C131 cells were prepared 28 h after infection, and those from mock-infected (lane 5) or Ad12-infected (lane 6) HeLa cells were prepared 20 h after infection. One-hundred-microgram amounts of protein were fractionated by electrophoresis on an SDS–10% polyacrylamide gel and analyzed for the presence of pTP or TP by Western blotting as described in Materials and Methods. The sizes of marker proteins are indicated on the left, and the positions of pTP and TP are indicated on the right. Ad12 virions (2 ⫻ 108 PFU) were coelectrophoresed as a control to identify the position of the Ad12 TP (lane 7).
as described above by using primers for the -actin gene (described above) and for the Ad12 DBP or the Ad12 pol gene: 12-DBP-f, 5⬘-GTAGTTCAAATTAA AAACGAC-3⬘; 12-DBP-r, 5⬘-TTAAAAATCAAATGGCTC-3⬘; 12-pol-f, 5⬘-AA ACATCAAATCCTCATC-3⬘; and 12-pol-r, 5⬘-CAAAGCCTCTGTAGCGTGG CC-3⬘. In some experiments, BHK21 cells overexpressing the Ad12 pTP, Ad12 E1A, or Ad2 E1A gene and infected with Ad12 were tested for the expression of late viral mRNAs. Total RNA was then prepared at 24 or 30 h p.i. and analyzed by RT-PCR with primers specific for the Ad12 fiber or for the Ad12 100K gene: 12-fiber-f, 5⬘-TGGTGAGCTCCGATGGGTTGG-3⬘; 12-fiber-r, 5⬘-TCCCCAC GAAGCTTGGGGAAC-3⬘; 12–100K-f, 5⬘-CAGATTCAAGCGGCGAAGGC C-3⬘; and 12–100K-r, 5⬘-GGAACCTTCCTCCTCCTCCTC-3⬘. RT products were amplified by 40 cycles as described above. Total RNA isolated from mockor Ad12-infected HeLa cells was used in control experiments. Immunoprecipitation of intracellular Ad12 DNA by Ad12-pTP antiserum. BHK21 cells were transfected with Ad12-pTP and pEGFP-C1, with Ad12-E1A and pEGFP-C1, or with Ad2-E1A and pEGFP-C1 as described above. At 18 h after electroporation, the BHK21 cells were infected with 100 PFU of Ad12 per cell, and human HeLa cells and hamster C131 cells were infected with 25 and 100 PFU per cell, respectively. Mock-infected BHK21 cells, nontransfected but Ad12-infected BHK21 cells, and Ad12-infected or mock-infected HeLa or C131 cells were investigated as controls. At 28 h p.i. or after mock infection, about 107 cells were suspended in ice-cold phosphate buffered saline (PBS), which contained 1% Igepal CA-630 (Sigma Chemicals), 0.1% SDS, and 4 mM Pefabloc SC protease inhibitor (Roche). Cells were disrupted by ultrasonic treatment for 2 min in a Branson B-12 sonifier. After the cellular debris had been removed, cell extracts were treated for 1 h at 4°C with 1.0 g of normal rabbit immunoglobulin G and 20 l of protein A-agarose conjugate (both from Santa Cruz Biotechnology). Subsequently, the beads were pelleted by centrifugation at 1,000 ⫻ g, and the precleared lysate was incubated at 4°C overnight under rotation with 15 l of Ad12-pTP rabbit antiserum and 30 l of protein A-agarose. The immunoprecipitates were collected by centrifugation for 5 min at 1,000 ⫻ g and washed four times with PBS. For the release of the Ad12 DNA from the Ad12 DNA pTP(TP)–Ad12 pTP antiserum–protein A–agarose complex, beads were resuspended in 50 l of 10 mM Tris-HCl (pH 8.0)–2 mM EDTA–10 mM NaCl–1% SDS and treated for 3 h at 55°C with 1 g of proteinase K per ml. DNA treated with proteinase K before immunoprecipitation was used as negative control. Agarose beads were removed by centrifugation, and the supernatant was analyzed for the presence of Ad12 DNA by a standard dot blot procedure. Briefly, DNA aliquots in the supernatant were treated with 0.4 M NaOH–5 mM EDTA, fixed on positively charged nylon membranes (Roche), and hybridized with 32 P-labeled Ad12 DNA as described above.
RESULTS Experimental design. In the present study, BHK21 cells were transfected with the pTP- or E1A-carrying constructs under HCMV promoter control. Upon electroporation, these Ad12 functions were overexpressed in BHK21 cells to a level that facilitated the de novo synthesis of unit-length Ad12 DNA after infection with Ad12. Ad12 DNA replication was assessed by labeling the newly synthesized Ad12 DNA with [3H]thymidine and by zone velocity sedimentation in alkaline sucrose gradients. The viral DNA peak fractions were further identified by Southern blot hybridization with 32P-labeled Ad12 DNA as probe. The newly synthesized Ad12 DNA was covalently linked to pTP as demonstrated by immunoprecipitation with an anti-Ad12 pTP serum. Minimal transcription of late viral genes in this complemented system was documented by RT-PCR. Synthesis of the late Ad12 fiber protein was not detectable by Western blot analyses. Reduced expression of pTP and E1A in Ad12-infected BHK21 cells. Since pTP plays a central role in the initiation of adenovirus DNA replication, the expression of Ad12-specific pTP was examined in productively or abortively Ad12-infected cells as well as in C131 hamster cells which can partly complement Ad12 DNA replication. A polyclonal antiserum raised against recombinant pTP of Ad12 was used in Western blot experiments to investigate pTP and TP syntheses in Ad12infected human or hamster cells (Fig. 1). In HeLa cells productively infected with Ad12, pTP was detected starting at 16 h p.i. (data not shown), and TP was detected starting at 20 h p.i. (Fig. 1, lane 6). In Ad12-infected C131 hamster cells, similar amounts of pTP were observed (Fig. 1, lane 4). Ad12 DNA can replicate to a limited extent in Ad12-infected C131 cells in which the Ad5-specific E1A and E1B functions are constitutively expressed and facilitate Ad12 DNA replication in the otherwise nonpermissive hamster cells
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FIG. 2. Minimal transcription of the E1A genes in Ad12-infected BHK21 cells. Total RNA (20 g) from mock-infected (lane 1) or Ad12-infected (lane 2) BHK21 cells or from mock-infected (lane 5) or Ad12-infected (lane 6) C131 cells was prepared at 24 h p.i., and that from mock-infected (lane 3) or Ad12-infected (lane 4) HeLa cells was prepared at 16 h p.i. The RNA was fractionated by electrophoresis on a 1% agarose–2.2 M formaldehyde gel and transferred to a nylon membrane. The RNA on the membrane was hybridized to a 32Plabeled E1A fragment of Ad12 DNA generated by PCR.
(14, 15, 25). However, Ad12 TP, which was present in Ad12 virions (Fig. 1, lane 7), was not produced in C131 cells (Fig. 1, lane 4). The lack of the Ad12 protease, a late viral protein, which is responsible for the proteolytic cleavage of pTP to TP, presumably explains the absence of the mature TP in nonpermisive hamster cells. In Ad12-infected BHK21 cells, pTP was made in amounts barely detectable and markedly smaller than those in Ad12-infected HeLa or C131 cells (Fig. 1, compare lane 2 to lanes 4 and 6), and TP was not detected. The concentrations of the Ad12 E1A and E1B proteins in Ad12-infected BHK21 cells were shown to be 6 to 20-fold lower than in Ad12-infected KB cells (20). We then investigated both the transcription of the Ad12 E1A genes and the synthesis of E1A proteins in Ad12-infected BHK1 cells as well
J. VIROL.
as in Ad12-infected C131 or HeLa cells. The results of RNA transfer (Northern blot) experiments revealed only minute amounts of E1A-specific transcripts in Ad12-infected BHK21 cells at 24 h p.i. (Fig. 2, lane 2). In Ad12-infected HeLa cells and in Ad12-infected C131 cells, E1A mRNAs were readily detectable at 16 and 24 h p.i., respectively (Fig. 2, lanes 4 and 6). No cross hybridization was observed between the RNA isolated from mock-infected C131 cells and the Ad12 E1Aspecific probe (Fig. 2, lane 5). As expected, the results of Western blot analyses revealed high expression levels of E1A in Ad12-infected C131 cells (Fig. 3, lane 4) or HeLa cells (Fig. 3, lane 6). Ad12 E1A-specific monoclonal antibodies did not react with the E1A proteins of Ad5 constitutively expressed in the mock-infected C131 complementing cell line (Fig. 3, lane 3). Hence, the E1A signal in Ad12-infected C131 cells could be unequivocally attributed to the Ad12-specific E1A protein. In contrast to Ad12-infected C131 or HeLa cells, Ad12-infected BHK21 cells produced only low levels of this protein (Fig. 3, lane 2). Based on these data, the question arose whether the overexpression of the Ad12 pTP or the Ad12 E1A genes in Ad12-infected BHK21 cells would help overcome the block of Ad12 DNA replication in this virus-cell system. The overexpression of Ad12 pTP, Ad12 E1A, or Ad2 E1A in BHK21 cells. BHK21 cells were transfected with one of the Ad12 pTP, Ad12 E1A, or Ad2 E1A expression constructs by electroporation as described in Materials and Methods. The pEGFP-C1 plasmid was cotransfected in all experiments to determine the efficiency of transfection. At 18 h after transfection by electroporation, cells were examined microscopically for the expression of GFP and then infected with Ad12. About 40% of the cells proved to be positively GFP transfected. At 28 h p.i. or post-mock infection (p.m.i.), BHK21 cells cotransfected with Ad12 pTP and pEGFP-C1, with Ad12 E1A and pEGFP-C1, or with Ad2 E1A and pEGFP-C1 were tested by Western blotting for the presence of pTP of Ad12, E1A of Ad12, or E1A of Ad2, respectively. After incubation with the
FIG. 3. E1A expression in Ad12-infected hamster or human cells. Protein extracts from mock-infected (lane 1) or Ad12-infected (lane 2) BHK21 cells or from mock-infected (lane 3) or Ad12-infected (lane 4) C131 cells were prepared at 24 h p.i., and those from mock-infected (lane 5) or Ad12-infected (lane 6) HeLa cells were prepared at 16 h p.i. Seventy-five-microgram amounts of protein were fractionated on an SDS–10% polyacrylamide gel and analyzed by Western blotting for the presence of the Ad12 E1A proteins. The sizes of the marker proteins and the gel positions of the Ad12 E1A proteins are indicated on the right. Unspecific reaction products different from the E1A proteins are indicated on the left.
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appropriate antibodies, membranes were stripped and subsequently reprobed with the polyclonal rabbit anti-GFP serum to monitor the efficiency of transfection and GFP expression. The expression of the Ad12 E1A proteins was analyzed by using a mouse monoclonal Ad12 E1A antibody. Western blot analyses revealed no E1A in mock-infected BHK21 cells (Fig. 4A, lane 1). In mock-transfected BHK21 cells infected with Ad12 or in cells transfected with pEGFP-C1 (vector control) and infected with Ad12, only tiny amounts of Ad12 E1A were detected (Fig. 4A, lanes 2 and 3). In BHK21 cells transfected with Ad12 pTP, the expression of Ad12 E1A-specific proteins was not increased (data not shown). In contrast, E1A expression was detectable in mock-infected or Ad12-infected BHK21 cells, which had previously been transfected with the Ad12 E1A construct (Fig. 4A, lanes 4 and 5). Expression of the cotransfected GFP gene was apparent in all transfection experiments (Fig. 4, lanes 3 to 5). Polyclonal antibodies against Ad2 E1A were used to examine the expression of the Ad2 E1A protein in BHK21 cells transfected with different constructs. This antiserum recognized specifically the E1A proteins of Ad2 or Ad5 and not those of Ad12 (data not shown). Western blot analyses revealed the expression of the Ad2 E1A protein in BHK21 cells transfected with the Ad2 E1A construct, although at levels lower than in Ad2-infected BHK21 cells or in Ad5-transformed C131 hamster cells (data not shown) which expressed the E1A of Ad5. The expression of Ad12 pTP and TP in transfected BHK21 cells was studied with polyclonal rabbit antibodies, which recognized both proteins. Western blot analyses revealed no pTP in mock-transfected and mock-infected BHK21 cells (Fig. 4B, lane 1), minimal amounts in pEGFP-C1-transfected and Ad12infected BHK21 cells (Fig. 4B, lane 2), and large amounts of pTP in extracts of Ad12-infected C131 cells (Fig. 4B, lane 8). Moreover, pTP synthesis was readily detected in BHK21 cells transfected with the Ad12-pTP and mock infected (Fig. 4B, lane 3) or infected with Ad12 (Fig. 4B, lane 4). The amounts of Ad12 pTP were increased in Ad12-infected BHK21 cells that had previously been transfected with the Ad12 E1A (Fig. 4B, lane 5) or Ad2 E1A (Fig. 4B, lane 6) construct, compared to the amounts in cells that had not been transfected with E1A, but had been infected with Ad12 (Fig. 4B, lane 2). The mature form of TP was not produced in any of these transfection experiments, but it was present in Ad12 virions (Fig. 4B, lane 9). Again, in all transfection experiments, the GFP gene, which was cotransfected as an internal control, was expressed (Fig. 4B, lanes 2 to 6). The nature of the additional protein band reacting unspecifically with the pTP antiserum (Fig. 4B, unspecific band) was not determined. The overexpression of the Ad12 pTP, Ad12 E1A, or Ad2 E1A construct in Ad12-infected BHK21 cells facilitates replication of Ad12 DNA. BHK21 cells grown on monolayers to halfconfluence were transfected by electroporation with one of the constructs Ad12 pTP, Ad12 E1A, or Ad2 E1A as described in Materials and Methods. In all experiments, the pEGFP-C1 plasmid was cotransfected. Transfection efficiency was monitored by UV light microscopy for green fluorescent cells and found to be about 40%. At 18 h after transfection, the cells were infected with CsCl-purified Ad12 at a multiplicity of 100 PFU per cell. At 14, 20, 28, and 36 h p.i., the total intracellular
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DNA was extracted, and then 10 g of that DNA was cleaved with EcoRI, blotted to a nylon membrane, and hybridized to the 32P-labeled 2,495-bp EcoRI-E and the 721-bp EcoRI-F fragments of Ad12 DNA (see EcoRI restriction map of Ad12 in Fig. 5), which lacked homologies to any of the transfected constructs. Hybridization signals were thus specific for the infecting Ad12 genome. Increases in signal intensities might indicate the de novo synthesis of Ad12 DNA. BHK21 cells transfected with the internal control plasmid pEGFP-C1 and subsequently infected with Ad12 failed to show any increase in Ad12-specific signals with time p.i. (Fig. 5, lanes a) as compared to mock-transfected and Ad12-infected BHK21 cells (Fig. 5, controls). In contrast, DNA from BHK21 cells transfected with the Ad12 pTP (Fig. 5, lanes b), Ad12 E1A (lanes c), or Ad2 E1A (lanes d) construct and then infected with Ad12 demonstrated increases in the intensities of the Ad12-specific EcoRI-E and EcoRI-F fragments starting at 20 h p.i. Phosphorimager analyses revealed increases by factors of 23 (Fig. 5, lanes b), 5 (lanes c), or 25 (lanes d) in comparison to the Ad12-infected BHK21 control cells (Fig. 5, lanes a and controls) at 28 h p.i. Ad12-specific signals increased up to 28 h p.i. and then diminished at 36 h p.i. At late times after infection, the concentrations of the overexpressed viral proteins might be reduced due to the degradation of the transfected DNA constructs. Thus, the concomitant degradation of newly synthesized Ad12 DNA might not be compensated for by continued Ad12 DNA replication. A shortage of additional viral and/or cellular functions, which are essential for Ad12 DNA replication, might also have played a role. In some experiments, BHK21 cells were cotransfected with both the Ad12 pTP and Ad12 E1A constructs or with the Ad12 pTP and Ad2 E1A constructs. These protocols did not improve the results in comparison to single transfection experiments and were therefore abandoned as possibly being too stressful for the cells. De novo synthesis of unit-length Ad12 DNA in nonpermissive BHK21 cells. The data presented in Fig. 5 suggested that the overexpression of the pTP or E1A genes of Ad12 facilitated Ad12 DNA replication in nonpermissive BHK21 hamster cells. The de novo synthesis of unit-length Ad12 DNA in BHK21 cells upon previous transfection with the Ad12 pTP, Ad12 E1A, or the Ad2 E1A construct was, therefore, investigated by labeling the newly synthesized DNA in these complemented systems with [3H]thymidine. Subsequently, the total intracellular DNA was analyzed by zone velocity sedimentation in alkaline sucrose density gradients upon alkali lysis of the cells at 28 h p.i. (4). In mock-infected BHK21, HeLa, or C131 cells (Fig. 6A, C, and D), Ad12 DNA was not synthesized, nor was it synthesized in BHK21 cells transfected previously with the pEGFP-C1 plasmid and then infected with Ad12 virions (Fig. 6B). The peaks of 3H-labeled DNA apparent in fractions 4 to 6 most likely represented cellular DNA synthesized during the labeling period between 6 and 28 h p.i. or p.m.i. In HeLa or C131 cells, which were competent for Ad12 DNA replication, Ad12 infection led to the synthesis of DNA that sedimented at fraction 17 or 15 (Fig. 6E and F, respectively). When nonpermissive BHK21 cells were first transfected with the Ad12 pTP (Fig. 6G), Ad12 E1A (Fig. 6H), or Ad2 E1A (Fig. 6I) construct and subsequently infected with Ad12 virions at 18 h posttransfection, a peak of newly synthesized, 3H-labeled DNA at fraction 16 was readily detected (Fig.
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FIG. 4. Overexpression of Ad12 E1A or Ad12 pTP in BHK21 cells. BHK21 cells were transfected with the Ad12 pTP, Ad12 E1A, or Ad2 E1A construct by electroporation as described in Materials and Methods. The pEGFP-C1 plasmid was cotransfected in all experiments as an internal control to monitor transfection and expression efficiencies. At 18 h after transfection, cells were infected with Ad12. At 28 h p.i., protein extracts were prepared, 100 g of protein was fractionated on an SDS–12.5% polyacrylamide gel, and the expression of Ad12 pTP or E1A was analyzed. (A) Western blot analyses of Ad12 E1A in protein extracts from mock-transfected and mock-infected BHK21 cells (lane 1), mock-transfected and Ad12-infected BHK21 cells (lane 2), BHK21 cells transfected with the pEGFP-C1 construct and infected with Ad12 (lane 3), and BHK21 cells transfected with the Ad12 E1A construct and mock (lane 4) or Ad12 (lane 5) infected. (B) Western blot analyses for the presence of the Ad12 pTP and TP in protein extracts from mock-transfected and mock-infected BHK21 cells (lane 1), BHK21 cells transfected with the pEGFP-C1 construct and infected with Ad12 (lane 2), BHK21 cells transfected with the Ad12 pTP construct and mock (lane 3) or Ad12 (lane 4) infected, BHK21 cells transfected with the Ad12 E1A (lane 5) or Ad2 E1A (lane 6) construct and infected with Ad12, and mock-infected (lane 7) or Ad12-infected (lane 8) C131 cells. Ad12 virions (4 ⫻ 108 PFU) were coelectrophoresed as a control to identify the gel position of the Ad12 TP (lane 9). For reprobing, the membranes in panels A and B were stripped and subsequently incubated with a polyclonal rabbit antibody raised against GFP to monitor the efficiency of transfection and GFP expression. The positions of E1A of Ad12 (A), pTP and TP of Ad12 (B), and GFP (A and B) are indicated on the right. Unspecific reaction products (A and B) of the polyclonal antibodies are designated on the left.
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FIG. 5. Southern blot analyses of the DNA from mock- or Ad12-infected BHK21 cells (controls) or from BHK21 cells transfected with the pEGFP-C1 (lanes a), Ad12 pTP (lanes b), Ad12 E1A (lanes c), or Ad2 E1A (lanes d) construct and infected with Ad12 at 18 h after transfection. At 14, 20, 28, and 36 h p.i., the total intracellular DNA was extracted, and 10 g of DNA was cleaved with EcoRI. The fragments were separated by electrophoresis on a 0.8% agarose gel, Southern blotted, and hybridized to the 32P-labeled EcoRI-E and EcoRI-F fragments of Ad12 DNA. DNA from mock- or Ad12-infected BHK21 cells (controls) was prepared at 28 h p.i. Purified Ad12 virion DNA (0.5 ng) was cleaved with EcoRI and coelectrophoresed as a control to identify the positions of the EcoRI-E and EcoRI-F fragments of Ad12 DNA. The EcoRI restriction map of Ad12 DNA is presented for orientation, and the positions of the E1A and pTP genes of Ad12 are designated by arrows.
6G to I). These peak fractions from each experiment were isolated and neutralized, and the DNA was ethanol precipitated. The DNA was subsequently identified as Ad12 DNA by electrophoresis on agarose gels, Southern blotting, and hybridization to 32P-labeled authentic Ad12 virion DNA (Fig. 7, lanes 1, 4, 7, and 10). Corresponding gradient fractions from mockinfected HeLa (Fig. 7, lane 2) or BHK21 cells (lane 13) contained no Ad12 DNA. DNA fraction 16 from nontransfected BHK21 cells or from BHK21 cells transfected with the pEGFP-C1 plasmid but infected with Ad12, which did not correspond to a 3H-labeled peak fraction from the sucrose gradients, showed a weak Ad12-specific signal, which most likely represented input (parental) Ad12 DNA (Fig. 7, lanes 14 and 12, respectively). Gradient fractions neighboring the 3Hlabeled peak fractions proved devoid of Ad12 DNA at the film exposure shown here (Fig. 7, lanes 3, 5, 6, 8, 9, and 11). The low levels of [3H[thymidine incorporation into cellular DNA in many of the experiments shown in Fig. 6 were probably due to the near confluent state of cells. Moreover, the transfection protocol might cause damage to the cellular DNA replication machinery. Upon Ad12 infection of permissive HeLa cells (Fig. 6E), only 20% of the number of cells used for the nonpermissive hamster cells (Fig. 6G to I) were lysed on top of the alkaline sucrose gradients. Hence, the viral DNA peak height in this experiment was at the level of complemented hamster cells. It is concluded that in BHK21 cells, which overexpress the pTP or the E1A genes of Ad12 or the E1A genes of Ad2, Ad12 DNA is capable of replicating to unit-length Ad12 DNA molecules. Apparently, in an unaided Ad12 infection of BHK21 cells, the levels of essential early viral proteins, E1A and pTP, do not suffice to support Ad12 DNA replication. The overexpression of the pTP gene of Ad12 alone elicits Ad12 DNA replication in this abortive cell system (Fig. 6G). Ad2 E1A overexpression seems to be somewhat more effective than the overexpression of Ad12 E1A in stimulating Ad12 DNA synthesis in BHK21 cells (compare Fig. 6H and I). The possibility has been considered that the incorporation of [3H]thymidine into full-length Ad12 DNA in E1A- or pTPtransfected BHK21 cells was due to repair synthesis. This in-
terpretation appears very unlikely, because a comparison of the data presented in Fig. 7, lanes 4, 7, and 10, demonstrated a substantial net increase in the amount of Ad12 DNA in the complemented systems (lanes 4, 7, and 10) compared to the trace amounts of parental Ad12 DNA detectable in the noncomplemented BHK21-Ad12 system (lanes 12 and 14) Immunoprecipitation of the newly synthesized Ad12 DNA with an anti-Ad12 pTP serum. Does the overexpressed Ad12 pTP in BHK21 cells actually function as the primer for Ad12 DNA replication and does it remain bound to the newly synthesized Ad12 DNA? BHK21 cells were transfected with one of the expression constructs and were then infected with Ad12 (described above). Immunoprecipitation of the pTP or TP protein presumably covalently bound to the newly synthesized Ad12 DNA was performed as described in Materials and Methods. HeLa or complementing C131 cells infected with Ad12 were used as controls. After the incubation of cell lysates with Ad12 pTP antiserum and protein A-agarose, the immunoprecipitates were treated with proteinase K to release the Ad12 DNA from the Ad12 DNA–pTP (or TP)–Ad12-pTP antiserum–protein A–agarose complex. The supernatants were then analyzed by Southern dot blot hybridization for the presence of Ad12 DNA by using 32P-labeled Ad12 DNA as probe. Substantial amounts of Ad12 DNA were immunoprecipitated by this procedure from Ad12-infected HeLa or C131 cells (Fig. 8B4 or B2, respectively). When pTP antiserum was not added to the extracts from Ad12-infected HeLa cells, an Ad12 DNAspecific signal was not obtained (Fig. 8B5). Ad12 DNA previously treated with proteinase K was not precipitable by this protocol (data not shown). Ad12 DNA could also be immunoprecipitated from nonpermissive BHK21 cells previously transfected with the Ad12 E1A, Ad2 E1A, or Ad12 pTP constructs and subsequently infected with Ad12 (Fig. 8A4, A5, and A6, respectively). The amounts of immunoprecipitated Ad12 DNA from BHK21 cells transfected with the Ad12 pTP or Ad2 E1A construct (Fig. 8A6 and A5, respectively) were comparable to those from Ad12-infected C131 cells (Fig. 8B2). Markedly less Ad12 DNA was immunoprecipitated from Ad12 E1A construct-transfected BHK21 cells (Fig. 8A4). These data corroborate the results on the de novo synthesis of Ad12 DNA
FIG. 6. Replication of full-length Ad12 DNA in BHK21 cells transfected with the Ad12 pTP, Ad12 E1A, or Ad2 E1A construct. BHK21 cells transfected with the pEGFP-C1 (B), Ad12 pTP (G), Ad12 E1A (H), or Ad2 E1A (I) construct were infected with Ad12 at 18 h after transfection. The newly synthesized DNA was labeled by adding 35 Ci of [3H]thymidine per ml of medium at 6 h p.i. The total intracellular DNA was analyzed by zone velocity sedimentation in alkaline sucrose density gradients 28 h after infection as described in Materials and Methods. DNA from mock-transfected and mock-infected BHK21 cells (A), from mock-infected (D) or Ad12-infected (F) C131 cells, or from mock-infected (C) or Ad12-infected (E) HeLa cells was prepared at 28 h p.i. and analyzed by the same protocol. 10048
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FIG. 7. Identification of Ad12 DNA in the peak gradient fractions. Peak 3H-labeled fractions from the alkaline sucrose density gradients shown in Fig. 6 synthesized in Ad12-infected HeLa cells (lane 1) or in Ad12-infected BHK21 cells that had previously been transfected with the Ad12 E1A (lane 4), Ad2 E1A (lane 7), or Ad12 pTP (lane 10) construct, were isolated, neutralized with acetic acid, ethanol precipitated, and analyzed by electrophoresis on a 0.7% agarose gel followed by Southern blotting. 32P-labeled Ad12 DNA was used as a hybridization probe. Corresponding fractions from mock-infected HeLa (lane 2) or BHK21 (lane 13) cells, from untransfected, merely Ad12-infected BHK21 (lane 14) cells, or from pEGFP-C1-transfected and subsequently Ad12-infected BHK21 (lane 12) cells were coelectrophoresed. Purified Ad12 virion DNA was also coelectrophoresed to identify authentic Ad12 DNA (lane 15). The fractions preceding and following each of the peak fractions 16 in the groups designated on the figure as BHK21/Ad12-E1A/Ad12 (lanes 3 and 5), BHK21/Ad2-E1A/Ad12 (lanes 6 and 8), and BHK21/Ad12-pTP/Ad12 (lanes 9 and 11) were coelectrophoresed and shown to contain no Ad12 DNA detectable at the film exposure time used here.
in transfected BHK21 cells as documented by velocity sedimentation and Southern analyses (Fig. 5 to 7). BHK21 cells transfected with Ad12 pTP or the Ad2 E1A constructs and, to a lesser degree with the Ad12 E1A construct, and infected with Ad12 were capable of de novo synthesis of Ad12 DNA. In mock-infected cells, Ad12 DNA was not detectable (Fig. 8A1, B1, and B3). Although the presence of parental Ad12 DNA could be documented by Southern blotting in merely Ad12-infected BHK21 cells or in BHK21 cells transfected with the pEGFP-C1 vector and subsequently infected with Ad12 (Fig. 5 and 7), we could not detect any Ad12 DNA in protein extracts isolated from these cells by immunoprecipitation (Fig. 8A2 and A3). The parental TP was likely degraded at 28 h p.i. Moreover, Ad12 pTP is produced in only tiny amounts in abortively infected BHK21 cells. The data presented in Fig. 8 demonstrate that pTP expressed in pTP- or E1A-transfected and Ad12-infected BHK21 cells is covalently bound to the newly synthesized Ad12 DNA and functions in the initiation of viral DNA replication. Similar conclusions hold for Ad12 infections in the complementing system of C131 cells. In addition, the data conclusively argue against the possibility of repair synthesis of Ad12 DNA in the Ad12 pTP, Ad12 E1A-, or Ad2 E1A-transfected and Ad12infected BHK21 cells. Enhanced transcription of the Ad12 pTP gene in Ad12infected BHK21 cells that overexpress E1A proteins of Ad12 or Ad2. The amount of the Ad12 pTP protein in Ad12-infected
BHK21 cells was markedly enhanced upon transfection with the Ad12 E1A or the Ad2 E1A construct (Fig. 4B, lanes 5 and 6). Does the overexpression of the E1A gene of Ad12 or of Ad2 stimulate the transcription of the Ad12 pTP gene in Ad12infected BHK21 cells? BHK21 cells were transfected with the Ad12 pTP, Ad12 E1A, or Ad2 E1A construct and subsequently infected with Ad12. The total cellular RNA was then analyzed by quantitative RT-PCR with specific primers for the Ad12 pTP and the control -actin genes. RNAs from mock- or pEGFP-C1-transfected and Ad12-infected BHK21 cells and RNAs from mockor Ad12-infected HeLa cells were used as controls. During PCR, the DNA was labeled with [32P]dCTP and subsequently fractionated by electrophoresis on a 4% polyacrylamide gel followed by phosphorimager analyses. The pTP-specific RTPCR products could first be detected after 35 PCR cycles when RNA from Ad12-infected BHK21 cells was transcribed into cDNA and amplified (data not shown). In mock- or pEGFPC1-transfected and Ad12-infected BHK21 cells, the intensity of the RT-PCR pTP-specific signals was about 25% of that in Ad12-infected HeLa cells (Fig. 9, compare lanes 2, 3, and 8). Upon transfection with the Ad12 pTP, Ad12 E1A, or Ad2 E1A construct followed by Ad12 infection, the intensities of the pTP signals were increased to 100, 48, and 80% (Fig. 9, lanes 4, 5, and 6, respectively) compared to that in productively Ad12infected HeLa cells (Fig. 9, lane 8). Apparently, the overexpression of the Ad12 or Ad2 E1A construct enhances the transcription of the pTP gene in Ad12-infected BHK21 cells.
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FIG. 8. Immunoprecipitation of Ad12 DNA by an anti-Ad12 pTP serum. Protein extracts from mock-infected BHK21 (A1), C131 (B1), or HeLa (B3) cells, from Ad12-infected BHK21 (A2), C131 (B2), or HeLa (B4) cells or from Ad12-infected BHK21 cells that had previously been transfected with the pEGFP-C1 (A3), Ad12 E1A (A4), Ad2 E1A (A5), or Ad12 pTP (A6) construct were immunoprecipitated with Ad12 pTP rabbit polyclonal antibodies together with the protein Aagarose conjugate as described in Materials and Methods. Protein extracts from Ad12-infected HeLa cells incubated with the protein A-agarose conjugate, but without addition of the Ad12 pTP antiserum (B5) or protein extracts from BHK21 cells transfected with Ad12 pTP and mock infected (B6) were negative controls. After proteinase K treatment, the supernatants were analyzed by Southern dot blot hybridization. 32P-labeled Ad12 DNA was used as a probe. Purified Ad12 DNA that had been dot blotted, but not subjected to immunoprecipitation, served as the positive control (C1).
The overexpression of the Ad2 E1A construct appears to be more effective in this respect. The transcription of the Ad12 DBP or the Ad12 pol gene remains unaltered in BHK21 cells transfected with the pTP or E1A gene and infected with Ad12 as compared with merely
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Ad12-infected BHK21 cells. In addition to Ad12 pTP, we have also analyzed by RT-PCR the transcription of the Ad12-specific replication genes, the Ad12 DNA binding protein (DBP), and the Ad12 DNA pol. BHK21 cells were transfected with the Ad12 pTP, Ad12 E1A, or Ad2 E1A construct and subsequently infected with Ad12. The total cellular RNA was analyzed by quantitative RT-PCR with specific primers for the Ad12 DBP and -actin genes (Fig. 10A) or for the Ad12 pol and -actin genes (Fig. 10B). RNAs from mock- or Ad12-infected BHK21, C131, or HeLa cells as well as RNAs from BHK21 cells transfected with the pEGFP-C1 and then infected with Ad12 were used as controls. Details of the procedure are described in Materials and Methods. The Ad12 DBP gene was transcribed abundantly at 24 and 30 h p.i. in all Ad12-infected cell types investigated. In particular, there was no difference in the transcription of the Ad12 DBP gene between Ad12-infected BHK21 cells, BHK21 cells overexpressing the Ad12 pTP or E1A gene or the Ad2 E1A gene and subsequently infected with Ad12, and in Ad12-infected C131 or HeLa cells (Fig. 10A). Similar results were obtained for the transcription of the Ad12 pol gene, except that transcription was low in all Ad12-infected cell types analyzed (Fig. 10B). The late Ad12 gene functions are not expressed in BHK21 cells that overexpress the pTP or E1A construct. In Ad12infected BHK21 cells transfected with Ad12 pTP, Ad12 E1A, or Ad2 E1A, pTP, but not the mature form of TP, is produced (Fig. 4B), probably due to the lack of the viral protease, which is a late viral gene product. We analyzed protein extracts from transfected and Ad12-infected BHK21 cells or from Ad12-
FIG. 9. Enhanced expression of the Ad12 pTP gene in Ad12-infected BHK21 cells that overexpress the E1A proteins of Ad12 or Ad2. BHK21 cells were mock transfected (lane 2) or transfected with the pEGFP-C1 (lane 3), Ad12 pTP (lane 4), Ad12 E1A (lane 5), orAd2 E1A (lane 6) construct and infected with Ad12 18 h after transfection. Total RNA was extracted at 24 h p.i. The total RNA from mock-infected BHK21 cells (lane 1) or from mock-infected (lane 7) or Ad12-infected (lane 8) HeLa cells was prepared at 24 h p.i. One-hundred-nanogram amounts of total RNA were analyzed by quantitative RT-PCR by using specific primers for the pTP gene of Ad12 and the cellular -actin gene as described in Materials and Methods. Portions of RT-PCR products were analyzed by electrophoresis on a 4% polyacrylamide gel followed by phosphorimager quantitation of the Ad12 pTP and -actin RT-PCR products. The sizes of DNA markers are indicated on the left, and the positions of the Ad12 pTP and -actin RT-PCR products are indicated on the right.
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FIG. 10. RT-PCR analyses of the transcription of the Ad12 DBP (A) or Ad12 pol (B) gene. BHK21 cells were transfected with the pEGFP-C1 (lane 3), Ad12 pTP (lane 4), Ad12 E1A (lane 5), or Ad2 E1A (lane 6) construct and infected with Ad12 18 h after transfection. The total RNAs from mock- and Ad12-infected BHK21 (lanes 1 and 2, respectively in panels A and B), C131 (lanes 7 and 8 in panel A, lanes 9 and 10 in B) or HeLa (lanes 9 and 10 in panel A, lanes 7 and 8 in panel B) cells as well as from transfected cells were prepared at 24 h p.i. One-hundred-nanogram amounts of RNA were subjected to quantitative RT-PCR by using specific primers for the Ad12 DBP and the cellular -actin genes (A) or for the Ad12 pol and -actin genes (B) as described in Materials and Methods. RT-PCR products were analyzed by electrophoresis on a 4% polyacrylamide gel followed by autoradiography and phosphorimager quantitation of the Ad12 DBP or pol and -actin RT-PCR products. The sizes of DNA markers are indicated on the left, and the positions of the Ad12 DBP, Ad12 pol, and -actin RT-PCR products are indicated on the right.
infected HeLa cells for the synthesis of the late viral fiber protein by Western blotting with an antiserum against the Ad12 fiber protein, which was readily detected with this assay in HeLa cells 28 h after productive infection with Ad12 (data not shown). In extracts of Ad12-infected BHK21 cells or C131 cells, as well as in all transfected and Ad12-infected BHK21
cells, the synthesis of the late fiber protein could not be demonstrated (data not shown). Even in the presence of Ad12 DNA replication, then, the synthesis of late viral proteins remained blocked in the abortive system. Late Ad12-specific RNA synthesis was investigated with the RT-PCR protocol by using specific primers for the L4 100K
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and L5 fiber genes. During productive adenovirus infection, these late genes, which were encoded in the L4 and L5 regions, were transcribed only late in the infection cycle after the first rounds of adenovirus DNA replication (12). Even after 40 cycles of PCR following RT, we could not detect Ad12 fiberspecific transcripts in BHK21 cells previously transfected with the Ad12 pTP, Ad12 E1A, or Ad2 E1A construct at 24 h p.i. with Ad12 (data not shown). At 30 h p.i., trace amounts of the fiber RT-PCR product were produced, when total RNA from Ad12-infected BHK21 cells overexpressing the Ad2 E1A gene was used. Similar results were obtained with RT-PCR for the Ad12 100K-specific RNA (data not shown). Only trace amounts of 100K RT-PCR products were detectable in BHK21 cells transfected with pTP or E1A genes and then infected with Ad12 at 30 h p.i. (data not shown). Apparently, the overproduction of the pTP or E1A gene of Ad12 or of the E1A gene of Ad2, which sufficed to elicit Ad12 DNA replication, failed to stimulate late Ad12 gene transcription in BHK21 cells at a significant level. DISCUSSION Although some of the early Ad12 genes are expressed in abortively Ad12-infected BHK21 Syrian hamster cells at low levels, viral DNA replication cannot be initiated. However, in the wake of the overexpression of the Ad12 pTP gene or, less markedly, of the Ad12 E1A gene upon transfection, Ad12 DNA replication becomes demonstrable 20 h after Ad12 infection of cells transfected with the HCMV promoter-driven constructs. Transfection with the control plasmid remains without effect on Ad12 DNA replication. Apparently, in merely Ad12-infected BHK21 cells, the levels of the E1A and/or pTP gene products lie below a critical threshold that has to be surpassed to facilitate viral DNA replication. In addition, the compartmental distribution and concentrations of these proteins at the nuclear sites of Ad12 DNA replication might remain insufficient for viral DNA replication to proceed. Obviously, pTP overexpression by itself, in the absence of high Ad12 E1A levels, is capable of triggering Ad12 DNA synthesis in the nonpermissive cell environment (Fig. 5 to 7). The overexpression of the transfected pTP gene does not increase the low levels of E1A protein in Ad12-infected BHK21 cells (data not shown). The initiation of Ad12 DNA replication in BHK21 cells, which overexpress Ad12 pTP, is unlikely to be due to the availability of sufficient levels of the E1A protein, which have not been increased in comparison to those in BHK21 cells that have been merely infected with Ad12. The overexpressed Ad12 pTP rather appears to have a decisive function in initiating and maintaining Ad12 DNA replication. It is conceivable, however, that the low levels of E1A protein available under these conditions, although below the threshold levels by themselves, cooperate with the above-threshold amounts of pTP and are thus capable of turning on Ad12 DNA replication directly or indirectly. Alternatively, Ad12 pTP may harbor new, previously unrecognized capacities to promote Ad12 DNA replication independently of functional E1A levels. It has been shown earlier that TP plays a role in the association of adenovirus DNA with the nuclear matrix of the cell (9, 24). Perhaps it is this aspect of Ad12 pTP function(s) that helps overcome the
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replication block of Ad12 DNA in a nonpermissive environment. BHK21 cells, which overexpress the E1A genes of Ad12 or of Ad2, produce sufficient amounts of Ad12 pTP upon Ad12 infection such that, in this system, Ad12 DNA of full length can be synthesized in moderate amounts (Fig. 6H and I, respectively). We propose that the overexpression of the E1A genes of Ad12 or Ad2 facilitates Ad12 DNA replication in nonpermissive BHK21 cells due to the activation of the Ad12 pTP gene. Of course, additional viral and cellular factors may also be involved in Ad12 DNA replication, and their production could also be influenced by pTP or E1A overexpression. The Ad12 DNA synthesized in this Ad12 pTP-complemented BHK21 cell system (Fig. 6G, H, and I) behaves in zone velocity sedimentation experiments like unit-length Ad12 DNA produced in the fully permissive HeLa cell system (Fig. 6E). Moreover, the newly synthesized viral DNA peak in BHK21 cells (Fig. 6G, H, and I) has been unequivocally identified as Ad12 DNA (Fig. 7). We have also shown that the de novo-synthesized Ad12 DNA in Ad12-infected BHK21 cells, which overexpress the Ad12 pTP, Ad12 E1A, or the Ad2 E1A construct, is bound to pTP (Fig. 8). This finding further documents the requirement for pTP as a primer for Ad12 DNA replication. Moreover, this experiment demonstrates true de novo synthesis of Ad12 DNA and eliminates the possibility of repair synthesis on parental Ad12 DNA in BHK21 hamster cells. Upon Ad12 infection of BHK21 cells, which overexpress the transfected Ad12 pTP or E1A gene or the Ad2 E1A gene, the transcription levels of the Ad12 DBP gene or the Ad12 pol gene do not differ from those in the merely Ad12-infected BHK21 cells. The DBP gene encoded by the E2A transcription unit is abundantly transcribed both in productively infected human cells and in all Ad12-infected hamster cells analyzed, irrespective of pTP and/or E1A levels. In contrast, the transcription of the E2B-encoded Ad12 pol gene is markedly reduced, even in the productive system. In Ad12-infected BHK21 cells, the E2B-encoded Ad12 pTP is only minimally expressed (Fig. 1). The expression levels of the Ad12 pol gene in mock- or in pTP- or E1A-transfected and Ad12-infected BHK21 cells remain low and are not significantly different from those in Ad12-infected C131 cells. At present, we cannot explain why the E2B-encoded Ad12 pTP and pol genes are much less actively transcribed than the E2A-encoded DBP gene. Overexpression of the Ad12 pTP gene in Ad12-infected BHK21 cells facilitates de novo Ad12 DNA synthesis in the BHK21 cell system, which has proved totally nonpermissive for Ad12 DNA replication unless aided by an above-threshold level of additionally supplied pTP from the E2B region of Ad12 DNA. In these cells, two other functions of the E2 transcription units, DBP (E2A) and Ad12 polymerase (E2B), are not augmented in transcriptional levels compared to merely Ad12-infected BHK21 cells (Fig. 10). These findings further support the conclusion that in Ad12-infected BHK21 cells, and possibly also in permissive human cells, Ad12 pTP can play an autonomous role in facilitating viral DNA replication. In hamster BHK297-C131 cells, which carry in an integrated form and constitutively express the E1A and E1B regions of
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Ad5 DNA (33), Ad12 DNA replication and late viral transcription proceed to a certain extent (14, 15). However, late viral proteins are not synthesized in these cells, although the fiber mRNA, which is produced in this complemented system, has been shown to have the authentic nucleotide sequence, leaders, and poly(A) tails (25). We have therefore proposed that, in addition to the transcriptional and replicational blocks for Ad12 DNA, the translation of late Ad12 mRNAs appears to be inhibited in hamster cells (25). In keeping with this interpretation, the minute amounts of late Ad12 mRNA synthesized in pTP- or E1A-transfected and Ad12-infected cells also fail to be translated into detectable amounts of late proteins like the fiber polypeptide as described in Results. When comparing the results presented in this report with those previously published in the Ad5-transformed BHK297C131 cells, the latter system markedly differs with respect to the higher transcriptional levels of the late Ad12-specific RNAs (14, 15, 25). Of course, the BHK297-C131 cells provide a constant supply of the constitutively transcribed E1 functions of Ad5 DNA, whereas the transfection of the E1A- or pTPcarrying plasmids seems to allow only the transient expression of limited amounts of the essential gene products. As stated above, in the transfection experiments, only trace amounts of late Ad12 RNAs are detectable. Perhaps, the transiently expressed Ad12 pTP, Ad12 E1A, or Ad2 E1 functions are degraded late after infection and/or are no longer functional to stimulate the late transcription of Ad12 DNA. Although unable to convert the BHK21-Ad12 system to a truly productive cycle, we recognize the necessity in this system to safeguard effectively against viral replication at many echelons to facilitate viral DNA integration and oncogenic cell transformation, and thus to secure the long-term, transgenerational persistence of the Ad12 genome in hamster cells. The integrated Ad12 genome in transformed hamster cells or in Ad12-induced tumor cells becomes de novo methylated in specific patterns (for review, see reference 7). ACKNOWLEDGMENTS We thank P. Freimuth, Brookhaven National Laboratory, Upton, N.Y., for providing rabbit anti-Ad12 fiber serum and R. Grand, University of Birmingham, Birmingham, United Kingdom, for a gift of monoclonal antibodies against Ad12 E1A. We are indebted to Petra Bo ¨hm for expert editorial work. This research was supported by the Deutsche Forschungsgemeinschaft through grants DO165/17–1 and SFB274-A1 and by the Wilhelm Sander Stiftung, Munich, Germany. REFERENCES 1. Bablanian, R., H. J. Eggers, and I. Tamm. 1965. Studies on the mechanism of poliovirus-induced cell damage. I. The relation between poliovirus-induced metabolic and morphological alterations in cultured cells. Virology 26:100–113. 2. Berk, A. J., F. Lee, T. Harrison, J. Williams, and P. A. Sharp. 1979. Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell 17:935–944. 3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. 4. Burger, H., and W. Doerfler. 1974. Intracellular forms of adenovirus DNA. III. Integration of the DNA of adenovirus type 2 into host DNA in productively infected cells. J. Virol. 13:975–992. 5. Chowrira, B. M., and L. A. Lucher. 1990. Extracts of hamster cells abortively infected with human adenovirus type 12 are competent to support initiation of viral DNA replication. Virology 176:289–291. 6. Doerfler, W. 1969. Nonproductive infection of baby hamster kidney cells (BHK21) with adenovirus type 12. Virology 38:587–606.
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