Viral gene products in adenovirus type-2 transformed hamster cells.

I have analyzed viral gene products expressed in five adenovirus type 2 (Ad2)- cytoplasmic, viral RNA which was selected by hybridization to cloned restriction endonuclease fragments of Ad2 DNA. Proteins synthesized in vitro were analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and compared with those directed by RNAs prepared from productively infected cells. The early regions E1 and E4 of adenovirus type 2 (Ad2) were found to be expressed in all of five Ad2-transformed hamster embryo cells lines. RNA transcribed from early region E2, which codes for the 72,000-molecular-weight (72K) DNA-binding protein was detected in cell line HE1 only, and early region E3 was expressed exclusively in cell line HE4. RNA transcribed from the region between approximately 12 and 35 map units, coding for immediate early (13.5K, 52/53K) and immediate early proteins (13.6K, 16K, 17K, 87K), as well as RNA from late genes, was not found in any of the cell lines HE1 to HE5 had electrophoretic mobilities similar to those programmed by RNA from productively infected cells.     

Methylation of adenovirus genes in transformed cells and in vitro: influence on the regulation of gene expression?

An inverse correlation has been described between the levels of DNA methylation in specific segments of adenovirus DNA integrated into the genomes of transformed and tumor cells and the extent to which these segments are expressed as messenger RNA. In the adenovirus type 2 (Ad2)-transformed hamster cell lines HE2 and HE3, the virus-specific DNA binding protein (DBP) is not expressed, and the DNA in the DBP gene is completely methylated in all 5'-CCGG-3' sites. At least part of the late promoter/leader sequence of the DBP gene is present in cell lines HE2 and HE3. In line HE1, on the other hand, the DBP is expressed, and the DNA in the DBP gene is unmethylated at the 5'-CCGG-3' (HpaII) sites. The late promotor/leader sequence of the DBP gene is expressed in cytoplasmic RNA isolated from line HE1. The effect of DNA methylation has also been tested in vitro in a microinjection system using Xenopus laevis oocytes. Unmethylated DNA fragments of Ad2 (E2a region) have been found to serve as active templates. When the same fragments are methylated at the 5'-CCGG-3' sites by the HpaII DNA-methyltransferase, viral RNA synthesis is inhibited upon microinjection into oocyte nuclei. These results provide direct evidence for the notion that DNA methylated at highly specific sites is somehow involved in the regulation of gene expression.     

Low molecular weight RNAs with homologies to cellular DNA at sites of adenovirus DNA insertion in hamster or mouse cells.

The adenovirus type 2 (Ad2)-transformed hamster cell line HE5 contains one or very few integrated copies of Ad2 DNA. At the site of insertion of Ad2 DNA, the cellular DNA sequence has been completely preserved and has homologies to small unpolyadenylated, cytoplasmic RNAs of 300 nucleotides in length and to minority populations of smaller RNAs present in HE5 cells and in normal hamster cells. The 300-nucleotide RNA is present on average in approximately 20 copies per cell. This RNA, and shorter RNAs, reveal homologies to the hamster DNA sequence of approximately 400 nucleotides to the right of the site of insertion of Ad2 DNA, which is present in one or very few copies per genome. The nucleotide sequence of the DNA segment homologous to this RNA does not contain open reading frames in excess of a sequence encoding 18 amino acids. Thus, it is unlikely that the small RNAs are actually translated and their function is unknown. The nucleotide sequence does not exhibit similarities to known low mol. wt. RNAs of eukaryotic origin. The low mol. wt. cellular RNA has been found in HE5 cells, in other hamster cell lines and organs, and also in mouse cells. There are differences with respect to size and abundance in the RNAs smaller than 300 nucleotides between HE5 cells and LSH hamster embryo cells. The adenovirus type 12 (Ad12)-induced mouse tumor CBA-12-1-T carries greater than 30 copies of integrated Ad12 DNA. The cellular DNA sequence at the site of Ad12 DNA insertion exhibits homologies to small RNAs (approximately 300 nucleotides long) from mouse cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation.

In the adenovirus type 2 (Ad2)-transformed hamster cell line HE3, the integrated late E2A promoter of Ad2 DNA is inactive, is methylated at all three 5'-CCGG-3' sequences, and can be reactivated by growing the cells in the presence of 50 microM 5-azacytidine (5-azaC). The three 5'-CCGG-3' sequences then become demethylated. Demethylation and reactivation are stable over 30 passages even after the removal of 5-azaC. The dormant late E2A promoter in cell line HE3 can also be reactivated by transfecting the cells with recombinant plasmids that carry the left terminal E1A and part of the E1B region of Ad2 DNA or the E1A 13S cDNA, but not with plasmids containing the E1A 12S cDNA. The E1A 13S cDNA encodes the 289-amino-acid trans-activating protein of Ad2. The E1A-mediated reactivation of the late E2A promoter is not accompanied by its demethylation in both DNA complements. Cell line HE3 produces constitutively E1A-encoded mRNAs and reactivates the methylated late E2A promoter-chloramphenicol acetyltransferase gene construct after transfection into HE3 cells. Constitutive levels of the endogenous E1A gene products in HE3 cells are detectable but, paradoxically, appear insufficient to reactivate the endogenous, chromosomally integrated E2A gene.     

Lack of evidence for methylation of parental and newly synthesized adenovirus type 2 DNA in productive infections.

Methylations of highly specific sites in the promoter and 5' regions of eucaryotic genes have been shown to shut off gene activity and thus play a role in the long-term regulation of gene expression. It was therefore of interest to investigate whether site-specific DNA methylations could also play a role in adenovirus DNA in productive infections. It has been reported earlier that adenovirion DNA is not detectably methylated (U. Günthert, M. Schweiger, M. Stupp, and W. Doerfler, Proc. Natl. Acad. Sci. USA 73:3923-3927, 1976). In the present study, evidence for the methylation of cytidine residues in 5'-CCGG-3' and 5'-GCGC-3' sequences or the methylation of adenine residues in 5'-GATC-3' and 5'-TCGA-3' sequences in intranuclear adenovirus type 2(Ad2) DNA isolated and analyzed early (5 h) or late (24 h) after infection could not be obtained. In Ad2 DNA, 22.5% of all 5'-CG-3' dinucleotides reside in 5'-CCGG-3' and 5'-GCGC-3' sequences. Intranuclear viral DNA was examined by restriction endonuclease cleavage by using HpaII, MspI, HhaI, DpnI, or TaqI and Southern blot hybridizations. The HindIII fragments of Ad2 DNA served as hybridization probes. The data rendered it very unlikely that free intracellular adenovirus DNA in productively infected cells was extensively methylated. Thus, DNA methylation was not a likely element in the regulation of free adenovirus DNA expression in productively infected cells.     

Viral DNA sequences and gene products in hamster cells transformed by adenovirus type 2.

Complementary strand-specific adenovirus DNA of full length or from endonuclease BamHI fragments was used as a probe to estimate the fractional representation and abundance of viral sequences in five hamster cell lines (Ad2HE1-5) transformed with UV-inactivated adenovirus type 2. The fraction of the viral genome present in the five transformed cell lines varied from 44% in the Ad2HE5 cell line to 84% in the Ad2HE3 cell line. The number of viral DNA copies per diploid cell equivalent ranged from 1.8 in the Ad2HE1 line to 7.1 in the Ad2HE4 line. In vivo labeling with [35S]methionine followed by immunoprecipitation with an antiserum against adenovirus type 2 early proteins revealed virus-specific polypeptides with molecular weights of 42,000 to 58,000 in extracts from all five hamster cell lines. Several other early viral polypeptides were detected in some of the adenovirus type 2-transformed hamster cell lines.     

The unmethylated state of the promoter/leader and 5'-regions of integrated adenovirus genes correlates with gene expression.

An inverse correlation has been established between the levels of DNA methylation at 5'-CCGG-3' (MspI/HpaII) sites in specific genes of integrated viral DNA in adenovirus type 12 (Ad12)-transformed hamster cell lines and the extent to which these genes are expressed ( Sutter and Doerfler , 1979, 1980). In general, early genes are transcribed into mRNA, while late genes are permanently switched off in these cell lines. Adenovirus type 2 genes methylated in vitro at 5'-CCGG-3' sites are not transcribed upon microinjection into nuclei of Xenopus laevis oocytes - unmethylated genes are expressed ( Vardimon et al., 1982a ). The MspI sites in the early and in some of the late Ad12 genes in cell lines HA12 /7, T637 , and A2497 -3 have now been precisely mapped. The data presented here reveal that the promoter/leader and 5'-regions of the early genes are unmethylated both at MspI sites and at 5'-GCGC-3' (HhaI) sites. In some instances, e.g., in the E2a regions in all three lines, the main parts of the early genes are partly methylated, even though the genes are expressed. In cell line HA12 /7, the early region E3 is not expressed, and the promoter/leader and 5'-regions of this segment are fully methylated. All late regions are completely methylated. The results suggest that the state of methylation in the promoter/leader and 5'-regions of integrated adenovirus genes is important in the control of gene expression.     

Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells.

The establishment of de novo-generated patterns of DNA methylation is characterized by the gradual spreading of DNA methylation (I. Kuhlmann and W. Doerfler, J. Virol. 47:631-636, 1983; M. Toth, U. Lichtenberg, and W. Doerfler, Proc. Natl. Acad. Sci. USA 86:3728-3732, 1989; M. Toth, U. Müller, and W. Doerfler J. Mol. Biol. 214:673-683, 1990). We have used integrated adenovirus type 12 (Ad12) genomes in hamster tumor cells as a model system to study the mechanism of de novo DNA methylation. Ad12 induces tumors in neonate hamsters, and the viral DNA is integrated into the hamster genome, usually nearly intact and in an orientation that is colinear with that of the virion genome. The integrated Ad12 DNA in the tumor cells is weakly methylated at the 5'-CCGG-3' sequences. These sequences appear to be a reliable indicator for the state of methylation in mammalian DNA. Upon explantation of the tumor cells into culture medium, DNA methylation at 5'-CCGG-3' sequences gradually spreads across the integrated viral genomes with increasing passage numbers of cells in culture. Methylation is reproducibly initiated in the region between 30 and 75 map units on the integrated viral genome and progresses from there in either direction on the genome. Eventually, the genome is strongly methylated, except for the terminal 2 to 5% on either end, which remains hypomethylated. Similar observations have been made with tumor cell lines with different sites of Ad12 DNA integration. In contrast, the levels of DNA methylation do not seem to change after tumor cell explanation in several segments of hamster cell DNA of the unique or repetitive type. Restriction (HpaII) and Southern blot experiments were performed with selected cloned hamster cellular DNA probes. The data suggest that in the integrated foreign DNA, there exist nucleotide sequences or structures or chromatin arrangements that can be preferentially recognized by the system responsible for de novo DNA methylation in mammalian cells.     

Purification and characterization of an early protein (E14K) from adenovirus type 2-infected cells.

One adenovirus type 2 (Ad2) early protein, with an apparent molecular weight of 14,000 in sodium dodecyl sulfate-polyacrylamide gels (E14K), was purified to homogeneity. Purification involved fractionation of cytoplasmic extracts, precipitation at low pH, and DEAE-cellulose, phosphocellulose, and hydroxylapatite chromatography. The yield was around 12 microgram of purified protein per 10(9) HeLa cells. The two Ad2 DNA binding proteins with molecular weights of 75,000 and 45,000 (E75K and E45K) were purified by the same procedure. Tryptic peptide analyses indicated that the E14K protein is unrelated to the DNA binding proteins. The purified E14K protein has a high content of basic amino acids and a sedimentation coefficient of 5.5S in the native state, corresponding to a molecular weight of around 95,000. Pulse-chase experiments suggest that the E14K polypeptide is a primary translation product. Immunoprecipitation with a monospecific antiserum against the E14K protein revealed that it is exclusively localized in the cytoplasm of infected cells. E14K started to be synthesized at 2 hpostinfection, with a maximal rate of synthesis at 4 to 6 h postinfection. Immunoprecipitation of cell extracts from four different Ad2-transformed hamster embryo cell lines revealed that only one (Ad2HE4) of them expresses this protein. The adenovirus-simian virus 40 hybrid virus (Ad2ND1) does not express this protein, suggesting that the gene for the E14K protein is located in the part of the Ad2 genome which is deleted in this hybrid virus.     

In vitro translation of adenovirus type 12-specific mRNA isolated from infected and transformed cells.

The early and late gene products of human adenovirus type 12 (Ad12), as well as the viral proteins synthesized in an Ad12-transformed cell line, were identified by translation of viral mRNA in an in vitro protein-synthesizing system. Cytoplasmic RNA was isolated from permissive KB or nonpermissive BHK cells infected with Ad12 and from Ad12-transformed HA12/7 cells. Virus-specific RNA was selected by hybridization to Ad12 DNA covalently bound to cellulose. Viral RNA was then translated in a fractionated rabbit reticulocyte cell-free system or in wheat germ S-30 extracts. The proteins synthesized were characterized by immunoprecipitation and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. RNA prepared from KB cells late after infection with Ad12 elicited the synthesis of most of the structural polypeptides of the virion and at least two presumably nonstructural Ad12 proteins. When viral RNA isolated early after infection of KB cells with Ad12 was translated in vitro, 10 polypeptides were observed: E-68K, E-50K, E-42K, E-39K, E-34K, E-21K, E-19K, E-13K, E-12K, and E-10K. Ad12-specific RNA was also isolated from the Ad12-transformed hamster cell line HA12/7, which contains several copies of the Ad12 genome integrated in the host genome. The RNA codes for at least seven polypeptides with molecular weights very similar to those of the early viral proteins.     

Patterns of integration of viral DNA in adenovirus type 2-transformed hamster cells

The patterns of integration of viral DNA in five lines of adenovirus type 2-transformed hamster cells have been investigated. Cell lines HE1 to HE5 were obtained by in vitro transformation of hamster embryo cells by ultraviolet light-inactivated Ad2². In all lines, segments in the central parts of the viral genome are missing. The lines HE1, HE2, HE3, HE4 and HE5 contain 2 to 4, 2 to 4, 6 to 10, about 10, and 2 to 3 genome fragment equivalents per cell, respectively.The patterns of integration in lines HE2 and HE3 are identical; however, the viral genome has been amplified in these cell lines to different extents. This result provides evidence for the post-integrational amplification of inserted viral genomes. It is also conceivable that line HE2 may have undergone losses of integrated Ad2 genomes. The persisting Ad2 genomes in lines HE2 and HE3 have deletions in parts of the EcoRI F and D fragments. The remainders of these fragments are linked to cellular DNA. The termini of the segments of the viral genome have been inverted and linked to each other. This linkage could have occurred via a circular intermediate in integration or via tandemly integrated viral genomes with subsequent deletion events. The linkage of the termini of viral DNA might be mediated by short sequences of cellular DNA.In line HE5, approximately 40% of the Ad2 genome is deleted, and the truncated segments, again comprising the terminal Ad2 DNA fragments, have been fused. The termini of the viral DNA are linked to cellular DNA. In lines HE1 and HE4 complex deletion and fusion events have altered the inserted Ad2 genomes.     

Revertants of Adenovirus Type 12-Transformed Hamster Cell Line T637 as Tools in the Analysis of Integration Patterns

Spontaneously arising morphological revertants of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 had been previously isolated, and it had been demonstrated that in these revertants varying amounts of the integrated Ad12 genome were eliminated from the host genome. In this report, the patterns of persistence of the viral genome in the revertants were analyzed in detail. In some of the revertant cell lines, F10, TR3, and TR7, all copies of Ad12 DNA integrated in line T637 were lost. In lines TR1, -2, -4 to -6, -8 to -10, and -13 to -16, only the right-hand portion of one Ad12 genome was preserved; it consisted of the intact right segment of Ad12 DNA and was integrated at the same site as in line T637. In revertant lines G12, TR11, and TR12, one Ad12 DNA and varying parts of a second viral DNA molecule persisted in the host genome. These patterns of persistence of Ad12 DNA molecules in different revertants supported a model for a mode of integration of Ad12 DNA in T637 hamster cells in which multiple (20 to 22) copies of the entire Ad12 DNA were serially arranged, separated from each other by stretches of cellular DNA. The occurrence of such revertants demonstrated that foreign DNA sequences could not only be acquired but could also be lost from eucaryotic genomes. There was very little, if any, expression of Ad12-specific DNA sequences in the revertant lines TR7 and TR12. Moreover, Ad12 DNA sequences which were found to be undermethylated in line T637 were completely methylated in the revertant cell lines G12, TR11, TR12, and TR2. These findings were consistent with the absence of T antigen from the revertant lines reported earlier. Hence it was conceivable that the expression of integrated viral DNA sequences was somehow dependent on their positions in the cellular genome. In cell line TR637, the early segments of Ad12 DNA were expressed and undermethylated; conversely, in the revertant lines G12, TR11, TR12, and TR2, the same segments appeared to be expressed to a limited extent and were strongly methylated.     

Integrated adenovirus type 12 DNA in the transformed hamster cell line T637: sequence arrangements at the termini of viral DNA and mode of amplification.

Approximately 20 to 22 copies of adenovirus type 12 (Ad12) DNA per cell were integrated into the genome of the cell line T637. Only a few of these copies seemed to remain intact and colinear with virion DNA. All other persisting viral genomes exhibited deletions or inversions or both in the right-hand part of Ad12 DNA. Spontaneously arising morphological revertants of T637 cells has lost viral DNA. In most of the revertant cell lines only the intact or a part of the intact viral genome was preserved; other revertant cell lines has lost all viral DNA. In three other Ad12-transformed hamster cell lines, HA12/7, A2497-3, and CLAC3 (Stabel et al., J. Virol. 36:22-40, 1980), major rearrangements at the right end of the integrated Ad12 DNA were not found. These studies were performed to investigate the phenomena of amplification, rearrangements, and deletions of Ad12 DNA in hamster cells.     

Evidence for an Adenovirus Type 2-Coded Early Glycoprotein

We have identified an adenovirus type 2 (Ad2)-induced early glycopolypeptide with an apparent molecular weight of 20,000 to 21,000 (20/21K), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 20/21K polypeptide could be labeled in vivo with [(3)H]glucosamine. [(35)S]methionine- and [(3)H]-glucosamine-labeled 20/21K polypeptides bound to concanavalin A-Sepharose columns and were eluted with 0.2 M methyl-alpha-d-mannoside. The pulse-labeled polypeptide appeared as a sharp band with an apparent molecular weight of 21K, but after a chase it converted to multiple bands with an average molecular weight of 20K. This variability in electrophoretic mobility is consistent with glycosylation or deglycosylation of the 20/21K polypeptide. Analysis of the pulse and pulse-chase-labeled forms by using partial proteolysis indicated that the polypeptides were highly related chemically, but not identical. Most of the 20/21K polypeptide is localized in the cytoplasm fraction of infected cells lysed by Nonidet P-40. The 20/21K polypeptide and a 44K polypeptide, labeled with [(35)S]methionine or [(3)H]glucosamine in Ad2-infected human cells, were precipitated by a rat antiserum against an Ad2-transformed rat cell line (T2C4), but not by antisera against three other Ad2-transformed rat cell lines, or by serum from nonimmune rats. The partial proteolysis patterns of the 20/21K and the 44K polypeptides were indistinguishable, indicating that the two polypeptides are highly related, and suggesting that the 44K polypeptide might be a dimer of the 20/21K polypeptide. The 20/21K polypeptide was also induced in Ad2-early infected monkey and hamster cells. These results imply that the 20/21K polypeptide is synthesized in Ad2-infected human, monkey, and hamster cells, and in one but not all Ad2-transformed rat cells. Thus, the 20/21K polypeptide is probably viral coded rather than cell coded and viral induced.