Ad2 DNA Research Articles

Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.

Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.

A novel method for transfection and expression of reconstituted DNA-protein complexes in eukaryotic cells.

Transfection of foreign DNA into eukaryotic cells has become an important tool in molecular biology. Based on the results of previous studies of the core structure of human adenoviruses, we have developed a novel transfection method. The procedure involves the in vitro reconstitution of foreign DNA-of viral or other origins-with the major core protein VII of adenovirus type 2 (Ad2) or protamine from salmon sperm. Both proteins are rich in basic amino acids and appear to share structural features. The DNA-protein complexes are added directly to the medium of eukaryotic cells. The in vitro formation of specific DNA-protein complexes can be assessed by gel electrophoretic analyses. Bovine serum albumin does not enter into specific complexes with DNA. Transfection of DNA-protein VII or DNA-protamine complexes results in their rapid transport into the cell nuclei. About 2-4 hr after transfection, up to 40% of the DNA added to cell cultures in complexes can be found in the nucleus, as compared with less than 10% of the DNA when other transfection methods are applied or when naked DNA is added to cell cultures. DNAs transfected by the new method into mammalian or insect cells retain their characteristic restriction patterns at least 48 hr after transfection and are expressed efficiently. Supercoiled circular plasmid DNAs are converted to open circular or linear DNA. Expression has been measured both for transiently expressed genes (chloramphenicol acetyltransferase gene, Ad2 DNA in human HeLa cells) and for genes that have been integrated into the host genome and are expressed permanently, such as the gene for neomycin phosphotransferase in hamster BHK21 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional interactions of the domains of the adenovirus DNA binding protein

The 34-kDa fragment of the carboxyl end of the adenovirus (Ad) DNA binding protein (DBP) binds to single-stranded (ss) DNA and is able to replace the intact 72-kDa DBP needed for Ad DNA replication in vitro. A similar fragment prepared from the temperature-sensitive (ts) mutant, H5ts107, which has a single amino acid change in the carboxyl end of the DBP, is temperature sensitive for DNA replication and defective in binding to ssDNA. However, in 20 m M NaCl which is the salt concentration during Ad DNA replication in vitro, the intact 72-kDa H5ts107 DBP is defective only in replication but not binding to DNA at nonpermissive temperatures. These observations indicate that the amino domain of the H5ts107 DBP can stabilize the binding of its carboxyl end to DNA.

Contactpoint analysis of the HeLa nuclear factor I recognition site reveals symmetrical binding at one side of the DNA helix.

Nuclear factor I (NFI) is a HeLa sequence-specific DNA-binding protein that is required for initiation of adenovirus (Ad) DNA replication and may be involved in the expression of several cellular genes. The interaction between NFI and its binding site on the Ad2 origin has been studied. Methylation interference and protection, u.v. irradiation of 5-BrdU substituted DNA and ethylation interference revealed major groove contacts with G and T, and phosphate backbone contacts. Computer stereographics show that the contacts are located in two blocks showing dyad symmetry to each other and 22 out of 23 contacts are accessible from one side of the helix. Inversion of the NFI binding site did not change the NFI dependent stimulation of Ad2 DNA replication in a reconstituted system. All data are compatible with NFI binding as a dimer at one side of the DNA helix.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

High affinity binding site for nuclear factor I next to the hepatitis B virus S gene promoter.

The hepatitis B virus (HBV) surface antigen (HBsAG) is encoded by the S gene under the regulation of a promoter in the pre-S1 region. The S gene promoter does not contain a 'TATA' box-like sequence, but there is a sequence resembling, in part, the late promoter of Simian virus 40 (SV40). In an attempt to study the regulation of the S gene promoter we looked for cellular proteins which bind to this region. We report here that a nuclear protein is tightly bound to the HBV genome at a position approximately 190 bases upstream from the S gene promoter. Evidence is provided to show that (a) this nuclear protein is the nuclear factor I (NF-I) that was previously found to be bound to the inverted terminal repeat of the adenovirus (Ad) DNA and to enhance Ad DNA replication in vitro and (b) this NF-I binding site is required for optimal activity of the S gene promoter.

Unusual DNA structures in the adenovirus genome.

More than 80% (approximately 29 kilobase pairs) of the adenovirus serotype 2 genome was surveyed for the presence of unusual DNA conformations. Seven recombinant DNAs containing the largest HindIII fragments of AD2 DNA were analyzed for the presence of negative supercoil-dependent S1 nuclease-sensitive sites. Four plasmids each contained a specific site of S1 nuclease sensitivity whereas the other three showed no reaction. Further investigation was focused on a plasmid containing one of the positively reacting fragments (fragment C) which contained the major late promoter at coordinate 16.4 on the genome; three serotypes (Ad2, Ad7, Ad12) were studied. Fine mapping studies revealed the S1-sensitive sites to be a small region (approximately 6 base pairs) located at the TATA box of the major late promoter in all three cases. Other determinations (supercoil relaxation, T7 gene 3 product sensitivity, bromoacetaldehyde reactivity, anomalous gel mobility, the influence of negative superhelical density on nuclease sensitivity) led to the conclusion that the B-helix deformation was not due to a previously recognized DNA conformation (left-handed Z-DNA, cruciform, bent DNA), but may be accounted for by the homopurine X homopyrimidine nature of this region.

Characterization of an adenovirus type 5 mutant carrying embedded inverted terminal repeats

During construction of an adenovirus type 5 (Ad5) deletion mutant, d1E1,3, lacking E1 and E3 sequences, we isolated a variant, d1E1,3-1, which had a direct repeat of viral DNA terminal sequences attached to the left end of the genome. Analysis of this variant with restriction enzymes and by hybridization of Southern blots with specific probes indicated that the extra terminal segment contained the left 2.6% (920 bp) of Ad5 joined to 352 bp of pBR322 which in turn was linked to the left end (minus 21 bp) of d1E1,3. During replication of d1E1,3-1 the extra terminal segment was found to transfer to the right end of the genome resulting in a second variant, d1E1,3-2, with duplicated terminal sequences at both ends of the viral genome. D1E1,3-2 in turn was shown to revert back to d1E1,3-1 at high frequency. Although evidence was obtained indicating that the extra segment could be lost from the left end, spontaneous mutants which had lost direct repeats from both ends were never isolated. It was, however, possible to remove the extra terminal repeat of d1E1,3-1 by cleavage with a restriction enzyme and to isolate d1E1,3 containing wt termini. The rearrangements occurring during replication of d1E1,3-1 and d1E1,3-2 may be consequences of the mode of replication of Ad5 DNA and bear some resemblance to segment inversion in herpesviruses.

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates.

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.

Chromosomal localization of integrated adenovirus DNA in productively infected and in transformed mammalian cells

The in situ hybridization technique has been used to localize adenovirus type 12 (Ad12) genomes on specific chromosomes of Ad12-transformed hamster cells as well as on specific chromosomes of human cells productively infected with Ad12. Hamster cell lines T637 and A2497-2 contain 20 to 22 and 17 copies of Ad12 DNA, respectively, in an integrated form. The results of in situ hybridization experiments demonstrate that the Ad12 DNA molecules are predominantly located on chromosomes 2, 7, 11, to 13, and 15 in cell line T637, and on chromosomes 7, 20, and perhaps 16 to 19 in cell line A2497-2. These data further document that viral DNA is chromosomally integrated and does not persist in a huge extrachromosomal, episomal structure. There is evidence from previous work that adenovirus DNA can also be covalently linked to human cellular DNA after productive adenovirus infection of human cells. In keeping with these earlier findings, we have now been able to show by in situ hybridization experiments that early in productive infection, i.e., 2 or 6 hr after infection, Ad12 genomes are predominantly associated with a limited number of human chromosomes. It appears biologically significant that these chromosomes, which belong to groups A and C II, have been the same ones in several independent infection experiments. Other chromosomes may also carry smaller amounts of viral DNA. In situ hybridization of Ad12 DNA to chromosomes of uninfected human cells yields no significant chromosomal association of viral DNA. The signal to noise ratio of grain counts over chromosomes from Ad12-infected cells to areas devoid of chromosomes is 5.6. The results presented raise the question of whether Ad12 DNA can integrate at selective sites in human chromosomes and whether this insertion event plays an essential role in early steps of viral transcription or replication. There is no direct evidence to suggest a biologic function for viral DNA integration in the productive infection cycle.

Adenovirus-2 early region IA protein synthesized in Escherichia coli extracts indirectly associates with DNA.

The interaction of adenovirus-2 (Ad2) early region IA (EIA) protein (encoded by the 13S mRNA) with DNA was examined using EIA protein synthesized in Escherichia coli extracts directed by a plasmid containing the cloned EIA gene. Without any purification, this protein when chromatographed over calf thymus DNA immobilized on cellulose, showed at least two types of salt-sensitive activities after associating with equal efficiency to both single- and double-stranded DNA; however, a putative C-terminal proteolytic fragment of the EIA protein (identified by immunoprecipitation with anti-serum specific to the EIA carboxy-terminus) showed 10-fold greater affinity to double- versus single-stranded DNA. When examined with Ad2 DNA, the EIA protein had a retention that was at least 2-fold higher compared to calf thymus DNA, suggesting some substrate specificity. It was also found that a 1.0 M salt concentration was required for the elution of the EIA protein from pBR322 DNA containing cloned regulatory sequences of adenovirus early regions II and III. This suggests that the strength of the protein interaction depends on the target DNA sequence. Finally, addition of uninfected HeLa cell extract to bacterial extracts containing EIA-like protein potentiated the association of the protein to double-stranded calf thymus DNA up to 7-fold. These data support the hypothesis that the EIA protein interacts with target DNA, presumably mediated by co-factor(s) in an indirect fashion.

Template requirements for in vivo replication of adenovirus DNA.

The adenovirus (Ad) DNA origin of replication was defined through an analysis of the DNA sequences necessary for the replication of plasmid DNAs with purified viral and cellular proteins. Results from several laboratories have shown that the origin consists of two functionally distinct domains: a 10-base-pair sequence present in the inverted terminal repetition (ITR) of all human serotypes and an adjacent sequence constituting the binding site for a cellular protein, nuclear factor I. To determine whether the same nucleotide sequences are necessary for origin function in vivo, we developed an assay for the replication of plasmid DNAs transfected into Ad5-infected cells. The assay is similar to that described by Hay et al. (J. Mol. Biol. 175:493-510, 1984). With this assay, plasmid DNA replication is dependent upon prior infection of cells with virus and only occurs with linear DNA molecules containing viral terminal sequences at each end. Replicated DNA is resistant to digestion with lambda-exonuclease, suggesting that a protein is covalently bound at both termini. A plasmid containing only the first 67 base pairs of the Ad2 ITR replicates as well as plasmids containing the entire ITR. Deletions or point mutations which reduce the binding of nuclear factor I to DNA in vitro reduce the efficiency of plasmid replication in vivo. A point mutation within the 10-base-pair conserved sequence has a similar effect upon replication. These results suggest that the two sequence domains of the Ad origin identified by in vitro studies are in fact important for viral DNA replication in infected cells. In addition, we found that two separate point mutations which lie outside these two sequence domains, and which have little or no effect upon DNA replication in vitro, also reduce the apparent efficiency of plasmid replication in vivo. Thus, there may be elements of the Ad DNA origin of replication which have not yet been identified by in vitro studies.

Template requirements for in vivo replication of adenovirus DNA.

The adenovirus (Ad) DNA origin of replication was defined through an analysis of the DNA sequences necessary for the replication of plasmid DNAs with purified viral and cellular proteins. Results from several laboratories have shown that the origin consists of two functionally distinct domains: a 10-base-pair sequence present in the inverted terminal repetition (ITR) of all human serotypes and an adjacent sequence constituting the binding site for a cellular protein, nuclear factor I. To determine whether the same nucleotide sequences are necessary for origin function in vivo, we developed an assay for the replication of plasmid DNAs transfected into Ad5-infected cells. The assay is similar to that described by Hay et al. (J. Mol. Biol. 175:493-510, 1984). With this assay, plasmid DNA replication is dependent upon prior infection of cells with virus and only occurs with linear DNA molecules containing viral terminal sequences at each end. Replicated DNA is resistant to digestion with lambda-exonuclease, suggesting that a protein is covalently bound at both termini. A plasmid containing only the first 67 base pairs of the Ad2 ITR replicates as well as plasmids containing the entire ITR. Deletions or point mutations which reduce the binding of nuclear factor I to DNA in vitro reduce the efficiency of plasmid replication in vivo. A point mutation within the 10-base-pair conserved sequence has a similar effect upon replication. These results suggest that the two sequence domains of the Ad origin identified by in vitro studies are in fact important for viral DNA replication in infected cells. In addition, we found that two separate point mutations which lie outside these two sequence domains, and which have little or no effect upon DNA replication in vitro, also reduce the apparent efficiency of plasmid replication in vivo. Thus, there may be elements of the Ad DNA origin of replication which have not yet been identified by in vitro studies.

Human cellular sequences detectable with adenovirus probes. I. Evidence for novel repeat sequences and a possible E1a-like cellular "gene".

Previous studies suggesting homology between human cellular DNA and the DNAs from adenovirus types 2 and 5 are extended in the present paper. A clone (ChAdh), isolated from a human genomic DNA library using an adenovirus probe, hybridized to discrete regions of adenovirus 2 DNA, including part of the transforming genes E1a and E1b, as well as to repeated sequences within human DNA. The E1a and E1b genes both hybridize to the same 300 base pair Sau3AI fragment within ChAdh although there is no obvious homology between E1a and E1b. The Ad 2 E1a gene was also used as a probe to screen other cellular DNAs to determine whether repeated sequences detectable with Ad2 DNA probes were conserved over long evolutionary periods. Hybridization was detected to the genomes of man, rat, mouse and fruit fly, but not to those of yeast and bacteria. In addition to a "smear" hybridization, discrete fragments were detected in both rodent and fruit fly DNAs. The experiments reported suggest the existence of two different types of cellular sequences detected by Ad 2 DNA: (1) repeated sequences conserved in a variety of eukaryote genomes and (2) a possible unique sequence detected with an E1a probe different from that responsible for hybridization to repeated sequences. This unique sequence was detected as an EcoRI fragment in mouse DNA and had a molecular size of about 8.8 kb.

N 6-methyldeoxyadenosine residues at specific sites decrease the activity of the E1A promoter of adenovirus type 12 DNA

The activity of eukaryotic promoters is highly sensitive to site-specific modifications by DNA methylations. We have used the E1A promoter of adenovirus type 12 (Ad12) DNA to investigate the effects of methylations at different promoter sites on its activity. The chloramphenicol acetyltransferase gene has served as an activity indicator. Activity of the E1A promoter is lost or markedly decreased by deoxycytidine methylation of two HpaII (5′-C-C-G-G-3′) or seven HhaI (5′-G-C-G-C-3′) sites upstream from the 3′ located T-A-T-A signal. There are two T-A-T-A signals in the E1A promoter of adenovirus type 12 DNA, one T-A-T-T-A-T sequence starting at nucleotide 276 (5′ located), a second T-A-T-T-T-A-A sequence starting at nucleotide 414 (3′ located). Deoxycytidine methylations at two AluI (5′-A-G-C-T-3′) sites downstream from the 5′ located T-A-T-A signal have no effect on promoter activity. When one EcoRI (5′-G-A-A-T-T-C-3′) or one TaqI (5′-T-C-G-A-3′) sequence at 281 base-pairs upstream or 61 base-pairs downstream from the 5′ located E1A T-A-T-A signal, respectively, is deoxyadenosine methylated, the promoter becomes inactive. Deoxyadenosine methylation at one MboI (5′-G-A-T-C-3′) site, which is located 127 nucleotides downstream from the 5′ located T-A-T-A signal, fails to decrease E1A promoter activity. There is no conspicuous anatomical relation of any of these sites to the two presumptive enhancer sequences in the E1A promoter. We conclude that 5-deoxymethylcytidine or N 6 -methyldeoxyadenosine residues have to be introduced at highly specific promoter sites to inactivate the promoter. These sites are probably different for different promoters.

Adenovirus DNA synthesis in Vitro is inhibited by the virus-coded major core protein

The effects of the adenovirus type 2 (Ad2) structural proteins on Ad DNA synthesis in vitro have been examined. Both of the viral core proteins, polypeptides V and VII were shown to inhibit Ad2 DNA synthesis in vitro; however, only the major core protein, polypeptide pII, inhibited DNA synthesis at a ratio of protein to DNA proportional to the number of polypeptide VII molecules associated with the Ad2 DNA in the mature virion. In addition, fractions containing the precursor to polypeptide VII, pVII, were capable of inhibiting Ad2 DNA replication in vitro to the same extent as polypeptide VII. Purified polypeptide VII bound to double-stranded DNA with no apparent sequence specificity. In addition, polypeptide VII protected Ad2 DNA from digestion with micrococcal nuclease. The binding of polypeptide VII was probably responsible for the inhibition of Ad2 DNA synthesis in vitro by virtue of rendering the DNA inaccessible to viral replication proteins. These results suggest that the core proteins must be removed from the Ad2 genome before the template can function in genome replication and that assembly of pVII on Ad2 DNA can terminate the replication process.

Formula omitted][formula omitted] transcription of a chromatin-like complex of major core protein VII and DNA of adenovirus serotype 2

Major core protein VII of adenovirus serotype 2 (Ad2) is thought to play a role as a histone octamer in eukaryotic cells. We compared the template activity of the VII-DNA complex formed in vitro with that of protein-free DNA. Hybridization assay of in vitro transcripts showed that transcription from regions located in the middle of Ad2 DNA decreased when Ad2 DNA formed a complex with VII. This suggests that the chromatin structure plays a role in regulation of transcription of the adenovirus genome.

Trans effect of the E1 region of adenoviruses on the expression of a prokaryotic gene in mammalian cells: resistance to 5' -CCGG- 3' methylation.

The plasmid construct pSVO-CAT has been used to test adenovirus promoter activities in the unmethylated or methylated state. We have now observed that the E2A late promoter of adenovirus type 2 (Ad2) DNA also activated the chloramphenicol acetyltransferase (CAT) gene upon transfection of the pAd2E2A-CAT construct into mammalian cells, and it was inactivated by specific methylations of three 5' -CCGG- 3' sites. Similar results had been reported previously after microinjecting promoter-methylated constructs into oocytes of Xenopus laevis. Surprisingly, it was found that the pSVO-CAT construct, which lacked eukaryotic promoter sequences, was able to express the CAT gene upon transfection into human or hamster cells that harbored and constitutively expressed the E1 region of Ad2 or Ad5 DNA. In these cells, the expression of the pAd2E2A-CAT construct was enhanced, but it was only partly sensitive to DNA methylation, possibly because DNA methylation was counteracted directly or indirectly by E1 functions. The pSVO-CAT construct was also expressed in HeLa or BHK21 cells upon cotransfection with a plasmid carrying the HindIII-G fragment of Ad2 DNA that contained the E1A region and part of the E1B region. By mapping pSVO-CAT-specific RNAs, we could demonstrate that pSVO-CAT activity in Ad2- or Ad5-transformed cells was mediated by prokaryotic promoter-like sequences in the pBR322 section of the construct, and it presumably functioned via trans-activation mediated by the E1 region. This trans-activation of pSVO-CAT in adenovirus-transformed cells was partly insensitive to DNA methylation.

Inhibition of protein synthesis enhances transformation of primary cells by viral DNA.

Inhibition of protein synthesis by cycloheximide after transfection and subsequent removal of the drug increased the transformation efficiency of primary cells by plasmids containing the left 4.5, 6.7, or 16% of the adenovirus (Ad) genome. The enhancement factor ranged from 2 to as much as 70 depending on the size of the viral DNA fragments used. Addition of cycloheximide before or at the time of transfection inhibited transformation, suggesting that viral protein synthesis is important during the early phase of transformation. Transient expression assays showed that cells treated with cycloheximide post-transfection contained as much as three times the amount of viral RNA transcribed from regions E1A and E1B. Conversion of a rat cell line lacking thymidine kinase activity (TK-) to the TK+ phenotype by a plasmid containing the herpes TK gene was severely inhibited by the drug treatment, suggesting that the enhancement effects of cycloheximide on transformation may be specific for Ad DNA. Cycloheximide treatment also increased the number of transformants induced by a transformation defective E1B mutant of Ad12 (cyt mutant). Plasmid containing only the E1A region of Ad12 transformed primary rat kidney cells with very low efficiency. The inclusion of E1B in the transfecting DNA fragments increased the transformation frequency by more than 400-fold, much higher than that achieved by cycloheximide. Thus, cycloheximide cannot replace E1B functions in transformation efficiency.