Restricted changes in the adenovirus DNA-binding protein that lead to extended host range or temperature-sensitive phenotypes

Human adenovirus fails to multiply efficiently in monkey cells owing to a block to late viral gene expression. Ad2hr400 through Ad2hr403 are a set of host range (hr) mutants which were selected for their ability to readily grow in these cells at 37 degrees C. The mutations responsible for this extended host range have previously been mapped to the 5' portion of the gene encoding the 72-kilodalton DNA-binding protein (DBP). DNA sequence analyses indicate that all four hr mutants contain the same alteration at coding triplet 130, which changes a histidine codon to a tyrosine codon. These results extend those of Anderson et al. (J. Virol. 48:31-39, 1983), which suggested that only this change in the DBP amino acid sequence can expand adenovirus host range to monkey cells. The hr phenotype does not appear to require phosphorylation of this tyrosine residue, since no phosphotyrosine was detected in DBP isolated from Ad2hr400-infected monkey cells. The hr mutants Ad2hr400 through Ad2hr403, however, are cold sensitive for growth in monkey cells. The mutant Ad2ts400, which was derived from Ad2hr400, represents a second class of hr mutants which can grow efficiently in monkey cells at 32.5 degrees C. The cold-resistant hr mutation of Ad2ts400 has previously been mapped to the 5' region of the DBP gene (map units 63.6 through 66). DNA sequence analysis of this region shows that this mutant contains the original hr alteration at coding triplet 130 as well as a second alteration at coding triplet 148, which changes an alanine codon to a valine codon. We suspect that the alterations at amino acids 130 and 148 change the structure of the amino-terminal domain of the DBP, allowing it to better interact with monkey cell components required for late viral gene expression. Ad2ts400 also contains a temperature-sensitive mutation which has previously been mapped to the 3' portion of the DBP gene (map units 61.3 through 63.6). Sequence analysis of this region indicates that the DBP coding triplet 413 has been altered. This change from a serine codon to a proline codon is the same alteration reported in the previously sequenced DBP mutants Ad5ts125 (W. Kruijer et al., Nucleic Acids Res. 9:4439-4457, 1981) and Ad5ts107 (W. Kruijer et al., Virology 124:425-433, 1983). Thus it appears that only a very limited number of changes in either the 5' or the 3' portion of the DBP gene can give rise to the hr or temperature-sensitive phenotypes, respectively.     

The function(s) provided by the adenovirus-specified, DNA-binding protein required for viral late gene expression is independent of the role of the protein in viral DNA replication

The adenovirus type 2 (Ad2) host range mutant Ad2hr400 grows efficiently in cultured monkey cells at 37 degrees C, but is cold sensitive for plaque formation and late gene expression at 32.5 degrees C. After nitrous acid mutagenesis of an Ad2hr400 stock, cold-resistant variants were selected in CV1 monkey cells at 32.5 degrees C. One such variant, Ad2ts400, was also temperature sensitive (ts) for growth in both CV1 and HeLa cells. Marker rescue analysis has been used to show that the two phenotypes, cold resistant and temperature sensitive, are due to two independent mutations, each of which resides in a different segment of the gene encoding the 72-kilodalton DNA binding protein (DBP). The cold-resistant mutation (map coordinates 63.6 to 66) is a host range alteration that enhances the ability of the virus to express late genes and grow productively in monkey cells at 32.5 degrees C. The temperature-sensitive mutation is in the same complementation group and maps to the same segment of the DBP gene (map coordinates 61.3 to 63.6) as the well-characterized DBP mutant Ad5ts125. Like Ad5ts125, Ad2ts400 is unable to replicate viral DNA or to properly shut off early mRNA expression at the nonpermissive temperature. Two sets of experiments with Ad2ts400 suggest that DBP contains separate functional domains. First, when CV1 cells are coinfected at the nonpermissive temperature with Ad2 plus Ad2ts400 (Ad2 allows DNA replication and entry into, but not completion of, the late phase of infection), normal late gene expression and productive growth occur. Second, temperature shift experiments show that, although DNA replication is severely restricted at the nonpermissive temperature in ts400-infected monkey cells, late gene expression occurs normally. These results indicate that the DBP activity required for normal late gene expression in monkey cells is functional even when the DBP's DNA replication activity is disrupted.     

Defective synthesis of early region 4 mRNAs during abortive adenovirus infections in monkey cells.

Human adenovirus 2 grows poorly in monkey cells, partly because of defects in late gene expression. Since deletions in early region 4 (E4) cause similar defects in late gene expression, we examined E4 mRNA expression in abortive infections. Processing of E4 mRNAs was defective during abortive infections, most likely at the level of splicing. At early times in productive infections in HeLa cells, the major E4 species produced is a 2-kb mRNA; at late times, a shift occurs so that smaller spliced E4 mRNAs are also produced. In CV-1 cells, a nonpermissive monkey cell line, this shift did not take place and only the 2-kb species was produced at late times, suggesting a defect in E4 mRNA splicing during abortive infections. The adenovirus DNA-binding protein (DBP) was required for normal processing of E4 mRNAs, since a host range mutant (hr602) containing an altered DBP gene showed a normal late E4 mRNA pattern in CV-1 cells; in addition, DBP was required during infections in HeLa cells for late E4 mRNA expression. DBP was not required for production of the late E4 pattern in transient expression assays in HeLa or 293 cells, suggesting that a second factor in addition to the DBP, present during infection but not transfection, modulates E4 mRNA processing.     

Adeno-associated virus helper activity of adenovirus DNA binding protein

The requirement for the adenovirus (Ad) single-stranded DNA binding protein (DBP) in the expression of adeno-associated virus (AAV) proteins was studied by specific immunofluorescent staining of infected cells and in vitro translation of RNA from infected cells. The Ad5 mutant ts125, which carries a mutation in the DBP gene, helped AAV as efficiently as the Ad5 wild type (WT) did at both the permissive (32 degrees C) and nonpermissive (40.5 degrees C) temperatures in HeLa and KB cells. Furthermore, at 40.5 degrees C ts125 was as efficient as Ad5WT was in inducing the expression of AAV proteins in a line of Detroit 6 cells which is latently infected with AAV. However, little if any AAV protein was synthesized when coinfections were carried out with Ad5WT in CV-C cells, a monkey cell line that is highly restrictive for human Ad replication unless the cells are also infected with simian virus 40. On the other hand, AAV protein was efficiently produced in CV-C cells in coinfections with the Ad5 mutant hr404, whose growth is unrestricted in CV-C cells and whose mutation also maps in the DBP gene. Finally, preparations of cytoplasmic RNA extracted from CV-C cells infected with AAV and Ad5WT or from CV-C cells infected with AAV, Ad5WT, and simian virus 40 were each capable of directing the in vitro synthesis of abundant amounts of AAV proteins in a rabbit reticulocyte lysate system. These results indicate that the abnormal DBP of ts125 still retains its helper function for AAV replication, but that the molecular feature of the DBP which relates to the monkey cell host range restriction of Ad's may also account for the observed block to AAV protein translation in CV-C cells.     

Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA

The adenovirus mutant Ad2ts111 has been previously shown to contain a mutation in the early region 2A gene encoding the single-stranded-DNA-binding protein that results in thermolabile replication of virus DNA and a mutation in early region 1 that causes degradation of intracellular DNA. A recombinant virus, Ad2cyt106, has been constructed which contains the Ad2ts111 early region 1 mutation and the wild-type early region 2A gene from adenovirus 5. This virus, like its parent Ad2ts111, has two temperature-independent phenotypes; first, it has the ability to cause an enhanced and unusual cytopathic effect on the host cell (cytocidal [cyt] phenotype) and second, it induces degradation of cell DNA (DNA degradation [deg] phenotype). The mutation responsible for these phenotypes is a single point mutation in the gene encoding the adenovirus early region 1B (E1B) 19,000-molecular-weight (19K) tumor antigen. This mutation causes a change from a serine to an asparagine in the 20th amino acid from the amino terminus of the protein. Three other mutants that affect the E1B 19K protein function have been examined. The mutants Ad2lp5 and Ad5dl337 have both the cytocidal and DNA degradation phenotypes (cyt deg), whereas Ad2lp3 has only the cytocidal phenotype and does not induce degradation of cell DNA (cyt deg+). Thus, the DNA degradation is not caused by the altered cell morphology. Furthermore, the mutant Ad5dl337 does not make any detectable E1B 19K protein product, suggesting that the absence of E1B 19K protein function is responsible for the mutant phenotypes. A fully functional E1B 19K protein is not absolutely required for lytic growth of adenovirus 2 in HeLa cells, and its involvement in transformation of nonpermissive cells to morphological variants is discussed.     

Two functions encoded by adenovirus early region 1A are responsible for the activation and repression of the DNA-binding protein gene.

Human adenovirus early region 1A (E1A) gene products differentially regulate the expression of early region 2A (E2A) encoding the DNA-binding protein (DBP). In a microinjection system, plasmids containing the DBP gene associated with both its early (map coordinate 75) and late (coordinate 72) promoters, or only with the early promoter, are inefficiently expressed, and the presence of E1A DNA is required for full expression. In contrast, the E2A plasmid in which the DBP gene is associated solely with its late promoter, efficiently produces DBP, the synthesis of which is significantly inhibited by an E1A gene product. To identify which of the E1A products is responsible for either activation or repression of DBP gene expression, two E1A mutants (Ad5hr1 and Ad2/5pm975) have been tested in the microinjection system in the presence of different DBP plasmids containing either one or both promoters. The results obtained indicate that the product encoded by the E1A 13S mRNA is responsible for the stimulation of DBP produced from the early promoter and that the 12S mRNA codes for the product which represses the synthesis of DBP from the late promoter. These results were confirmed using clones in which the E2A early or late promoter was associated to the chloramphenicol acetyltransferase (CAT) gene and assayed for CAT activity after cell transfection in the absence or in the presence of wild-type or mutant E1A plasmids, and we have also shown that this promoter-dependent regulation is reflected in the relative amount of specific DBP mRNA.     

Adenovirus type 12 E1A protein expressed in Escherichia coli is functional upon transfer by microinjection or protoplast fusion into mammalian cells.

We efficiently expressed, in Escherichia coli, and purified the protein product encoded by the human adenovirus type 12 (Ad12) 13S mRNA. The functional properties of the E1A protein were analyzed by introducing the protein by microinjection or protoplast fusion into living mammalian cells. We showed that the E. coli-expressed E1A protein induces gene expression of the adenovirus type 5 (Ad5) E1A deletion mutant Ad5dl312. The purified E1A protein rapidly and quantitatively localized to the cell nucleus after microinjection into the cytoplasm. In addition, we raised high-titered monospecific antibodies to the purified Ad12 E1A protein. Using deleted forms of an adenovirus type 2 and Ad5 hybrid (Ad2/5) E1A protein, we showed that all of the epitopes conserved between Ad2/5 E1A and Ad12 E1A protein that are recognized by the Ad12 E1A-specific antiserum map to within the first exon-encoded amino-terminal half of the protein.     

Mutations that allow human Ad2 and Ad5 to express late genes in monkey cells map in the viral gene encoding the 72K DNA binding protein.

Five host-range mutants (Ad2hr400–hr403, Ad5hr404) of human adenovirus serotype 2 and 5 (Ad2 and Ad5) which overcome the block to growth of wild-type adenovirus in monkey cells have been isolated. They form plaques and multiply efficiently in both monkey and human cells. The alteration in each of these mutants allows the full expression of all viral late genes, in marked contrast to the depressed synthesis of many late proteins in monkey cells infected with the parental Ad2 or Ad5. The altered gene encodes a diffusible product, since the mutation acts in trans to enhance the synthesis of wild-type Ad3 late proteins during co-infections of monkey cells with Ad2hr400 and Ad3. Restriction enzyme analysis of the genomes of all the host-range mutants show that none of them contain major alterations. In addition, an earlier report (Klessig and Hassell, 1978) indicated that Ad2hr400 does not contain SV40 sequences, which in some adenovirus-SV40 hybrid viruses allows efficient multiplication in monkey cells. The mutation responsible for the extended host range has been physically mapped by marker rescue experiments using isolated restriction enzyme fragments of the mutants to transfer the new phenotype to wild-type adenovirus. The alteration in each of the five mutants is located in a region (coordinates 62–70.7; coordinates 62–68 for Ad5hr404) which encodes predominantly the 72K DNA binding protein. More detailed mapping using Ad2hr400 fragments places the mutation (coordinates 62.9–65.6) entirely within the 72K gene. The multifunctional nature of the 72K protein and some of its similarities to SV40 T antigen are discussed.     

Control of Adenovirus Gene Expression: Cellular Gene Products Restrict Expression of Adenovirus Host Range Mutants in Nonpermissive Cells

Adenovirus type 5 (Ad5) host range mutants dl312 and hr-1, with lesions in region E1A (0 to 4.5 map units) of the viral genome, fail to accumulate virus-specific early RNA during infection in HeLa cells. In a recent report, we showed that the addition of anisomycin, a stringent inhibitor of protein synthesis, at 1 h after infection of HeLa cells with hr-1 virus resulted in the accumulation of properly spliced and translatable mRNA from all early regions (M. G. Katze, H. Persson, and L. Philipson, Mol. Cell. Biol. 1:807-813, 1981). Based on these results we proposed a model in which expression of early mutant RNA was achieved through inactivation of a cellular protein normally causing a reduction in the amount of viral RNA. These studies have been extended in the present report, which shows that early viral proteins can be detected in Ad5 dl312- and Ad5 hr-1-infected HeLa cells which have been treated for several hours with anisomycin either shortly after infection or before infection. A pulse of drug treatment also resulted in expression of substantial amounts of adenovirus structural proteins after infection with both Ad5 hr-1 and Ad5 dl312, whereas in drug-free controls no late proteins were detected. The Ad5 hr-1 virus previously reported to be DNA replication negative in nonpermissive HeLa cells was found to replicate its DNA, albeit at low levels, when anisomycin was present either from 1 to 5 h postinfection or for 5 h before infection. When infectious virus production was examined in mutant-infected cells the titer of Ad5 dl312 virus was found to increase at least 500-fold in anisomycin-treated HeLa cells. Taken together, these and our previous results suggest that the block in gene expression characteristic for complementation group I Ad5 host range mutants in HeLa cells can be overcome by inactivating cellular gene products serving as negative regulators of viral gene expression.     

Expression of the chloramphenicol acetyl transferase gene in human cells under the control of early adenovirus subgroup C promoters: effect of E1A gene products from other subgroups on gene expression

A hierarchy of dominance has been observed in HeLa cells co-infected with two serotypes of adenovirus belonging to different subgroups. DNA replication and late protein synthesis of one serotype are inhibited by those of the other. The degree of inhibitory effect has the following decreasing order: adenovirus type 3 (Ad3) and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad12 (A), Ad2 and Ad5 (C) [Delsert and D'Halluin, Virus Res. 1 (1984) 365-380]. HeLa cells were first transfected with recombinant plasmids carrying Ad5 E2A or E3 promoters fused to the chloramphenicol acetyl transferase gene (cat), and then infected with human Ad belonging to different subgroups. All the serotypes tested were found to be able to stimulate both E2A and E3 promoters. When HeLa cells were co-transfected with either of the previous plasmids, plus a second plasmid carrying the Ad3 E1A region, the same stimulatory effect was observed. However, an inhibitory effect on Ad5 E2A and E3 promoters seemed to occur when both Ad2 E1A (subgroup C) and Ad3 E1A (subgroup B) genes were present together. To determine which one of the early products was responsible for the observed repression effect, and to assign the target on the genome of subgroup C Ad, a plasmid was constructed in which the sequences at the 5' end of the Ad2 E1A region were fused to the structural sequences of the cat gene. In HeLa cells transfected with this plasmid, CAT activity was significantly increased after co-transfection with a plasmid carrying the Ad2 E1A region, but decreased with a plasmid carrying the Ad3 E1A region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells.

In soluble protein extracts obtained from adenovirus productively infected cells, monoclonal antibodies directed against the early region 1B 58,000-dalton (E1B-58K) protein immunoprecipitated, in addition to this protein, a polypeptide of 25,000 molecular weight. An analysis of tryptic peptides derived from this 25K protein demonstrated that it was unrelated to the E1B-58K protein. The tryptic peptide maps of the 25K protein produced in adenovirus 5 (Ad5)-infected HeLa cells and BHK cells were identical, whereas Ad3-infected HeLa cells produced a different 25K protein. The viral origin of this 25K protein was confirmed by an amino acid sequence determination of five methionine residues in two Ad2 tryptic peptides derived from the 25K protein. The positions of these methionine residues in the 25K protein were compared with the nucleotide sequence of Ad2 and uniquely mapped the gene for this protein to early region 4, subregion 6 of the viral genome. A mutant of Ad5 was obtained (Ad5 dl342) which failed to produce detectable levels of the E1B-58K protein. In HeLa cells infected with this mutant, monoclonal antibodies directed against the E1B-58K protein failed to detect the associated 25K protein. In 293 cells infected with Ad5 dl342, which contain an E1B-58K protein encoded by the integrated adenovirus genome, the mutant produced an E4-25K protein which associated with the E1B-58K protein derived from the integrated genome. Extracts of labeled Ad5 dl342-infected HeLa cells (E1B-58K-) were mixed in vitro with extracts of unlabeled Ad5 wild type-infected HeLa cells or 293 cells (E1B-58K+). When the mixed extracts were incubated with the E1B-58K monoclonal antibody, a labeled E4-25K protein was coimmunoprecipitated. When extracts of Ad5 dl342-infected HeLa cells and uninfected HeLa cells (both E1B-58K-) were mixed, the E1B-58K monoclonal antibody failed to immunoselect the E4-25K protein. These data provide evidence that the E1B-58K antigen is physically associated with an E4-25K protein in productively infected cells. This is the same E1B-58K protein that was previously shown to be associated with the cellular p53 antigen in adenovirus-transformed cells.     

Induction of gene expression by exon 2 of the major E1A proteins of adenovirus type 5.

We have constructed an adenovirus type 5 (Ad5) E1A mutant, dl1119/520, that produces essentially only exon 2 of the major E1A proteins. In infected primary baby rat kidney cells, this mutant induced expression of the E1B 55-kDa protein, and in infected human KB cells, it induced expression of this protein, the E2A 72-kDa protein, and hexon. In KB cells, this mutant grew substantially better than Ad5 dl312, which lacks E1A, and as well as Ad5 dl520, an E1A mutant producing only the 243-residue protein. These results suggest that exon 2 of E1A proteins on its own was able to activate gene expression. We also constructed mutants of dl1119/520, containing small deletions in regions of exon 2 that others found to be associated with effects on the properties of E1A transformants. None of these deletions destroyed gene activation completely, indicating that there may be some redundancy among sequences in exon 2 for inducing gene expression. The two deletions that decreased induction the most, residues 224 to 238 and 255 to 270, were in regions reported to be associated with the expression of a metalloprotease and with enhanced transformation, suggesting that exon 2 may regulate expression of genes governing cell growth. It is remarkable that all sections of E1A proteins, exon 1, the unique region, and exon 2, have now been found to affect gene expression.     

E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter.

The transformation-defective Vero cell host range mutant CS-1 of the highly oncogenic adenovirus type 12 (Ad12) (Ad12-CS-1) has a 69-bp deletion in the early region 1A (E1A) gene that removes the carboxy-terminal half of conserved region 2 and the amino-terminal half of the Ad12-specific so-called spacer that seems to play a pivotal role in the oncogenicity of the virus. Despite its deficiency in immortalizing and transforming primary rodent cells, we found that the E1A 13S protein of Ad12-CS-1 retains the ability to bind p105-RB, p107, and p130 in nuclear extract binding assays with glutathione S-transferase-E1A fusion proteins and Western blot analysis. Like wild-type E1A, the mutant protein was able to dissociate E2F from retinoblastoma-related protein-containing complexes, as judged from gel shift experiments with purified 12S and 13S proteins from transfection experiments with an E1A expression vector or from infection with the respective virus. Moreover, in transient expression assays, the 12S and 13S products of wild-type Ad12 and Ad12-CS-1 were shown to transactivate the Ad12 E1A promoter containing E2F-1 and E2F-5-motifs, respectively, in a comparable manner. The same results were obtained from transfection assays with the E2F motif-dependent E2 promoter of adenovirus type 5 or the human dihydrofolate reductase promoter. These data suggest that efficient infection by Ad12 and the correlated virus-induced reprogramming of the infected cells, including the induction of cell cycle-relevant mechanisms (e.g. E2F activation), can be uncoupled from the transformation properties of the virus.     

Conditional Lethal Mutants of Adenovirus 2-Simian Virus 40 Hybrids I. Host Range Mutants of Ad2+ND1

Human adenovirus type 2 (Ad2) grows poorly in monkey cells, although this defect can be overcome by co-infection with simian virus 40 (SV40). The nondefective Ad2-SV40 hybrid virus, Ad2(+)ND1, replicates efficiently in both human and African green monkey kidney cells, presumably due to the insertion of SV40 sequences into the Ad2 DNA. Several mutants of Ad2(+)ND1 have been isolated that grow and plaque poorly in monkey cells, although they retain the ability to replicate and plaque efficiently in HeLa cells. One of these mutants (H39) has been examined in detail. Studies comparing the DNA of the mutant with Ad2(+)ND1 either by the cleavage patterns produced by Escherichia coli R.RI restriction endonuclease digestion or by heteroduplexing reveal no differences. The pattern of protein synthesis of Ad2(+)ND1 and H39 in monkey cells is quite different with the mutant resembling Ad2, which is defective in the synthesis of late proteins. However, in human cells, the proteins synthesized by H39 and the parent Ad2(+)ND1 are very similar. The production of SV40 U antigen in H39-infected cells is different from that in Ad2(+)ND1-infected cells. Finally, the growth of H39 in monkey cells can be complemented by SV40.     

cyt gene of adenoviruses 2 and 5 is an oncogene for transforming function in early region E1B and encodes the E1B 19,000-molecular-weight polypeptide

A total of 59 cytocidal (cyt) mutants were isolated from adenovirus 2 (Ad2) and Ad5. In contrast to the small plaques and adenovirus type of cytopathic effects produced by wild-type cyt+ viruses, the cyt mutants produced large plaques, and the cytopathic effect was characterized by marked cellular destruction. cyt mutants were transformation defective in established rat 3Y1 cells. cyt+ revertants and cyt+ intragenic recombinants recovered fully the transforming ability of wild-type viruses. Thus, the cyt gene is an oncogene responsible for the transforming function of Ad2 and Ad5. Genetic mapping in which we used three Ad5 deletion mutants (dl312, dl313, and dl314) as reference deletions located the cyt gene between the 3' ends of the dl314 deletion (nucleotide 1,679) and the dl313 deletion (nucleotide 3,625) in region E1B. Restriction endonuclease mapping of these recombinants suggested that the cyt gene encodes the region E1B 19,000-molecular-weight (175R) polypeptide (nucleotides 1,711 to 2,236). This was confirmed by DNA sequencing of eight different cyt mutants. One of these mutants has a single missense mutant, two mutants have double missense mutations, and five mutants have nonsense mutations. Except for one mutant, these point mutations are not located in any other known region E1B gene. We conclude that the cyt gene codes for the E1B 19,000-molecular-weight (175R) polypeptide, that this polypeptide is required for morphological transformation of rat 3Y1 cells, and that simple amino acid substitutions in the protein can be sufficient to produce the cyt phenotype.     

Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs.

The studies described here demonstrate that the expression of many early adenovirus mRNAs is dependent upon the activity of a pre-early viral product. This viral gene product is defective in adenovirus 5 host range (Ad hr) group I mutants. Adenovirus 5 host range mutants were previously isolated by their ability to replicate in the adenovirus 5-transformed human embryonic cell line 293 and by their inability to replicate efficiently in HeLa cells (Harrison, Graham and Williams, 1977). The group I complementation class of host range mutants has been mapped by marker rescue between 0 and 4.4 units (Frost and Williams, 1978). We have used the S1 nuclease gel technique to examine the expression of early mRNA after infection of HeLa cells with Ad5 hr group I and II mutants. The Ad5 hr group II mutants stimulate the synthesis of a wild-type pattern of early mRNAs. In contrast, infection of HeLa cells with Ad5 hr group I mutants gives rise to only two early mRNAs. These mRNAs map from 1.5–4.4 units, or in the same region as the Ad5 hr group I mutations. Since infection of HeLa cells with Ad5 hr group I mutants was defective for synthesis of cytoplasmic mRNAs complementary to three early regions in the right half of the genome and to the early region 4.5–11.0 units, we also analyzed nuclear RNA from these cells by the S1 nuclease gel technique for the presence of precursor RNA chains. Nuclear precursors were not detected in Ad5 hr group I-infected HeLa cells, suggesting that the gene product defective in these mutants is required for synthesis of stable nuclear RNA from the three early regions in the right half of the genome and from the early region 4.5–11.0 units.     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

Homologous recombination of adenovirus DNA in mammalian cells: enhanced recombination following UV-irradiation of the virus.

We have used adenovirus as a molecular probe to examine the recombination of viral DNA following infection of mammalian cells. The technique gives a quantitative measure of homologous recombination between adenovirus type 2 (Ad2) and Ad5PyMTR3. Ad5PyMTR3 is an insertion mutant of Ad5 containing polyoma virus (Py) DNA inserted into a deleted E1 region of the Ad5 genome. Cells were coinfected with Ad2 and Ad5PyMTR3 and at an appropriate time after infection, viral DNA was extracted from the infected cells, digested with restriction endonuclease and electrophoresed through an agarose gel. Although Ad2 and Ad5 have more than 99% DNA homology, they differ sufficiently in their restriction endonuclease patterns, such that recombinant viral DNA molecules containing the Py insert could be detected and quantified by Southern blotting and hybridization to a radioactive Py DNA probe. Using this method we are able to detect and quantitate recombinant viral DNA molecules containing the Py insert which are present at frequencies down to at least 1 in 100. Recombination was detected in Chinese hamster ovary cells, monkey kidney cells, human HeLa cells, normal human fibroblasts and SV40 transformed human fibroblasts. In experiments using HeLa cells, the frequency of recombination between the Py insert on Ad5PyMTR3 and a number of unique restriction enzyme sites on Ad2 increased with the distance from the Py insert to the restriction site. Also in HeLa cells, recombination increased with increasing amounts of viral DNA synthesis and with increasing UV dose to the virus. UV-irradiation of both coinfecting viruses with 1500 J/m2 resulted in a more than 100-fold reduction in the amount of viral DNA synthesized and about a 3-fold increase in the frequency of recombination.     

Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein.

We have determined the nucleotide sequence of the gene encoding adenovirus type 2 (Ad2) DNA binding protein (DBP). From the nucleotide sequence the complete amino acid sequence of Ad2 DBP has been deduced. A comparison of the amino acid sequences of Ad2 and Ad5 DBP, both 529 residues long, reveals that the C-terminal 354 residues of both sequences are identical. Within the N-terminal 175 amino acid residues Ad2 and Ad5 show nine differences. The site of mutation in Ad2 ND1ts23, a mutant with a temperature-sensitive DNA replication, was mapped at the nucleotide level. A single nucleotide alteration in the DBP gene, resulting in a leucine leads to phenylalanine substitution at position 282 in the amino acid sequence is responsible for the temperature-sensitive character of this mutant. Previously, we localized the mutation of another DBP mutant with a temperature-sensitive DNA replication (H5ts125) at position 413 in the amino acid sequence of the DBP molecule (Nucleic Acids Res. 9 (1981) 4439-4457). These mapping data are discussed in relation to the structure and function of the DBP molecule.