Several E4 region functions influence mammary tumorigenesis by human adenovirus type 9

Among oncogenic adenoviruses, human adenovirus type 9 (Ad9) is unique in eliciting exclusively estrogen-dependent mammary tumors in rats and in not requiring viral E1 region transforming genes for tumorigenicity. Instead, studies with hybrid viruses generated between Ad9 and the closely related nontumorigenic virus Ad26 have roughly localized an Ad9 oncogenic determinant(s) to a segment of the viral E4 region containing open reading frame 1 (E4-ORF1), E4-ORF2, and part of E4-ORF3. Although subsequent findings have shown that E4-ORF1 codes for an oncoprotein essential for tumorigenesis by Ad9, it is not known whether other E4 region functions may similarly play a role in this process. We report here that new results with Ad9/Ad26 hybrid viruses demonstrated that the minimal essential Ad9 E4-region DNA sequences include portions of both E4-ORF1 and E4-ORF2. Investigations with Ad9 mutant viruses additionally showed that the E4-ORF1 protein and certain E4-ORF2 DNA sequences are necessary for Ad9-induced tumorigenesis, whereas the E4-ORF2 and E4-ORF3 proteins are not. In fact, the E4-ORF3 protein was found to antagonize this process. Also pertinent was that certain crucial nucleotide differences between Ad9 and Ad26 within E4-ORF1 and E4-ORF2 were found to be silent with respect to the amino acid sequences of the corresponding proteins. Furthermore, supporting a prominent role for the E4-ORF1 oncoprotein in Ad9-induced tumorigenesis, an E1 region-deficient Ad5 vector that expresses the Ad9 but not the Ad26 E4-ORF1 protein was tumorigenic in rats and, like Ad9, promoted solely mammary tumors. These findings argue that the E4-ORF1 oncoprotein is the major oncogenic determinant of Ad9 and that an undefined regulatory element(s) within the E4 region represents a previously unidentified second function likewise necessary for tumorigenesis by this virus.     

Human adenovirus type 9-induced rat mammary tumors.

Following subcutaneous inoculation of newborn Wistar-Furth rats with human adenovirus type 9 (Ad9), 16 of 16 female and 0 of 11 male rats developed mammary tumors. Tumor-positive animals usually developed tumors in multiple glands. Histopathological analyses indicated that three general categories of tumor could be identified. Mammary fibroadenomas were the most common tumor type encountered, but phyllodeslike tumors and solid sarcomas were also frequently found. In situ hybridization and immunohistochemical techniques established that benign fibroadenomas were derived from mammary fibroblasts (collagen type I- and vimentin-positive cells) and that malignant tumors were derived from myoepithelial cells (collagen type IV-, vimentin-, and muscle-specific actin-positive cells). The fact that mammary tumors were limited to female rats suggested that female hormones are essential for tumor growth and development. In this regard, ovariectomy of Ad9-infected female rats prevented tumor development, while subsequent diethylstilbestrol (DES) treatment elicited tumor formation. In addition, Ad9-infected and castrated male rats which received DES also developed mammary tumors. Established male mammary tumors regressed when DES treatment was stopped and reappeared after DES treatment was resumed. Together, these results indicate that estrogen is required for both initiation and maintenance of Ad9-induced mammary tumors. Southern blot analysis of high-molecular-weight tumor DNA showed that mammary tumor cells contained single or multiple integrated copies of the entire Ad9 genome. RNase protection experiments established that estrogen receptor as well as Ad9 E1a and E4 mRNAs were expressed in mammary tumors, but Ad9 E3 and, surprisingly, E1b mRNAs were not expressed at detectable levels.     

Gene organization of the transforming region of adenovirus type 7 DNA

The sequence of the leftmost 11% of the weakly oncogenic human adenovirus type 7 (Ad7) DNA has been determined. This part of the Ad7 viral genome encompasses early region E1 which has been shown to be involved in the process of cell transformation in vitro (Dijkema et al., 1979). From the nucleotide sequence and determined coordinates of the E1 mRNAs, we are able to predict the primary structure of the polypeptides encoded by the transforming region of Ad7. The organization of the E1 region of Ad7 and of other adenovirus serotypes (Bos et al. 1981) leads to the proposal of a novel mechanism for gene regulation at the translational level in which protein synthesis can initiate at either the first or the second AUG triplet available in mRNA. The differences between the large E1b-specific tumor antigens of adenovirus types 12, 7 and 5 may explain the differences in oncogenicity of these viruses.     

Oncogenicity by adenovirus is not determined by the transforming region only.

We have constructed a nondefective recombinant virus between the nononcogenic adenovirus 5 (Ad5) and the highly oncogenic Ad12. The recombinant genome consists essentially of Ad5 sequences, with the exception of the transforming early region 1 (E1) which is derived from Ad12. HeLa cells infected with the recombinant virus were shown to contain the Ad12-specific E1 proteins of 41 kilodaltons (E1a) and 19 and 54 kilodaltons (both encoded by E1b). The recombinant virus replicated efficiently in human embryonic kidney cells and HeLa cells, showing that the transforming regions of Ad5 and Ad12 had similar functions in productive infection. After the recombinant virus was injected into newborn hamsters, no tumors were produced during an observation period of 200 days. Thus, despite the fact that all products required for oncogenic transformation in vitro were derived from the highly oncogenic Ad12, the recombinant virus did not produce tumors in vivo. These data show that tumor induction by adenovirus virions is not determined only by the gene products of the transforming region.     

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.     

Complementation of human adenovirus type 5 ts mutants by human adenovirus type 12.

Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.     

Tn5 mutagenesis of the transforming genes of human adenovirus type 5

We have cloned the entire human adenovirus type 5 (Ad5) genome into the pBR322 plasmid in two segments: the BamHI-A fragment (21 kb) and the BamHI-B fragment (15 kb). We have also generated a series of clones with smaller Ad5 DNA inserts, all containing the left-end of the viral genome. One such clone, pXCl, containing the left 16% of the Ad5 DNA molecule, has been shown to transform rodent cells by DNA transfection. We have used the transposable element Tn5 as an insertion mutator to isolate pXCl mutants containing Tn 5 inserted at a large number of sites. By assaying transforming activity of selected pXC::Tn5 plasmids we have identified Ad5 sequences which are essential for DNA-mediated transformation. Our results with these mutants and with a plasmid pCDl, containing a deletion within the Ad5-transforming region, indicate that sequences present in both early region la and the N-terminal region of early region 1b are essential for DNA-mediated transformation.     

DNA damage sensors ATM, ATR, DNA-PKcs, and PARP-1 are dispensable for human immunodeficiency virus type 1 integration

Integration of a DNA copy of the viral RNA genome is a crucial step in the life cycle of human immunodeficiency virus type 1 (HIV-1) and other retroviruses. While the virally encoded integrase is key to this process, cellular factors yet to be characterized are suspected to participate in its completion. DNA damage sensors such as ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related), DNA-PK (DNA-dependent protein kinase), and PARP-1 [poly(ADP-ribose) polymerase 1] play central roles in responses to various forms of DNA injury and as such could facilitate HIV integration. To test this hypothesis, we examined the susceptibility to infection with wild-type HIV-1 and to transduction with a vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1-derived lentiviral vector of human cells stably expressing small interfering RNAs against ATM, ATR, and PARP-1. We found that integration normally occurred in these knockdown cells. Similarly, the VSV-G-pseudotyped HIV-1-based vector could effectively transduce ATM and PARP-1 knockout mouse cells as well as human cells deficient for DNA-PK. Finally, treatment of target cells with the ATM and ATR inhibitors caffeine and wortmannin was without effect in these infectivity assays. We conclude that the DNA repair enzymes ATM, ATR, DNA-PKcs, and PARP-1 are not essential for HIV-1 integration.     

A carboxy-terminal region required by the adenovirus type 9 E4 ORF1 oncoprotein for transformation mediates direct binding to cellular polypeptides.

Human adenovirus type 9 (Ad9) is unique among oncogenic adenoviruses in that it elicits exclusively mammary tumors in rats and requires the viral E4 region open reading frame 1 (9ORF1) gene for tumorigenicity. The 9ORF1 oncogenic determinant codes for a 14-kDa transforming protein, and three separate regions of this polypeptide, including one at the extreme C terminus, are necessary for transforming activity. In this study, we investigated whether the 9ORF1 transforming protein interacts with cellular factors. Following incubation with cell extracts, a glutathione S-transferase (GST)-9ORF1 fusion protein associated with several cellular phosphoproteins (p220, p180, p160, p155), whereas GST fusion proteins of transformation-defective 9ORF1 C-terminal mutants did not. Similar interactions requiring the 9ORF1 C terminus were revealed with protein-blotting assays, in which a GST-9ORF1 protein probe reacted specifically with cellular polypeptides having gel mobilities resembling those of the 9ORF1-associated cellular phosphoproteins, as well as with additional cellular polypeptides designated p140/p130. In addition, GST fusion proteins containing 9ORF1 C-terminal fragments associated with some of the 9ORF1-associated cellular polypeptides, as did GST fusion proteins of full-length wild-type Ad5 and Ad12 E4 ORF1 transforming proteins. Significantly, the results of coimmunoprecipitation analyses suggested that the same cellular polypeptides also associate with wild-type but not C-terminal-mutant 9ORF1 proteins in vivo. Together, these findings suggest that the 9ORF1 C terminus, which is essential for transformation, participates in specific and direct binding of the 9ORF1 oncoprotein to multiple cellular polypeptides. We propose that interactions with these cellular factors may be responsible, at least in part, for the transforming activity of the 9ORF1 viral oncoprotein.     

Abortive and transforming infection of rat cell line 3Y1 by adenovirus type 12.

Human adenovirus type 12 underwent an abortive and transforming infection in clonal cultures of rat cell line 3Y1. The frequency of transformation was proportional to the dose of input virus. The ratio of PFU to transforming units was 1.1 X 10(5).     

Control functions of adenovirus transformation region E1A gene products in rat and human cells.

Altered control of the rat cell cycle induced by adenovirus requires expression of transformation region E1A, but not of E1B, E2A, E2B, or late genes. We show here that neither E3 nor E4 is required, so the effect results directly from an E1A product. Mutants with defects in the 289-amino-acid (aa) E1A product had little or no effect on the rat cell cycle even at 1,000 IU per cell. A mutant (pm975) lacking the 243-aa E1A product altered cell cycle progression, but less efficiently than did wild-type virus. The 289-aa E1A protein is therefore essential for cell cycle effects; the 243-aa protein is also necessary for the full effect but cannot act alone. Mutants with altered 289-aa E1A proteins showed different extents of leak expression of viral early region E2A as the multiplicity was increased; each leaked more in human than in rat cells. dl312, with no E1A products, failed to produce E2A mRNA or protein at 1,000 IU per cell in rat cells but did so in some experiments in human cells. There appears to be a very strict dependence of viral early gene expression on E1A in rat cells, whereas dependence on E1A is more relaxed in HeLa cells, perhaps due to a cellular E1A-like function. Altered cell cycle control is more dependent on E1A function than is early viral gene expression.     

Lytic and transforming functions of individual products of the adenovirus E1A gene.

To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA E1A region but not in the presence of a 12S E1A cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the E1A 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.     

Adenovirus type 9 E4 open reading frame 1 encodes a transforming protein required for the production of mammary tumors in rats.

The E4 region of human adenovirus type 9 (Ad9) transforms established rat embryo fibroblasts and encodes an essential determinant for the production of estrogen-dependent mammary tumors in rats. Testing of the seven Ad9 E4 open reading frames (ORFs) individually for transformation of the established rat embryo fibroblast cell line CREF indicated that only Ad9 E4 ORF1 possessed a significant ability to generate transformed foci on these cells. In contrast, the E4 ORF1 sequences from human Ad5 and Ad12 lacked the transforming potential exhibited by Ad9 E4 ORF1. Cell lines derived from Ad9 E4 ORF1-transformed foci expressed the 14-kDa Ad9 E4 ORF1 protein and formed colonies in soft agar. In addition, the Ad9 E4 ORF1 protein was required for initiation of mammary oncogenesis in vivo, as E4 ORF1 mutant viruses failed while E4 ORF2 and ORF3 mutant viruses succeeded in eliciting mammary tumors in animals. A role for Ad9 E4 ORF1 in tumor maintenance was suggested by the fact that 100% of virus-induced mammary tumors expressed the E4 ORF1 protein. Taken together, the facts that the Ad9 E4 ORF1 protein exhibits transforming potential in culture and is required by Ad9 to produce mammary tumors in animals suggest that Ad9 E4 ORF1 is a new viral oncoprotein.     

Interferon induction by adenovirus type 12: stimulatory function of early region 1A.

Adenoviruses are generally weak interferon inducers, triggering chicken embryo fibroblast cells by a UV-resistant viral component, probably the capsid or capsid elements, to produce 50 to 100 IU of interferon per ml. Adenovirus types 12, 18, and 31, however, can induce by a UV-sensitive mechanism 10 to 20 times more interferon than other types do. By using mutant and recombinant adenoviruses, we demonstrated that early region 1A was responsible for the enhanced interferon production of chicken cells infected with adenovirus type 12.     

Two regions of the adenovirus early region 1A proteins are required for transformation.

Regions of the adenovirus type 5 early region 1A (E1A) proteins that are required for transformation were defined by using a series of deletion mutants. Deletion mutations collectively spanning the entire protein-coding region of E1A were constructed and assayed for their ability to cooperate with an activated ras oncogene to induce transformation in primary baby rat kidney cells. Two regions of E1A (amino acids 1 to 85 and 121 to 127) were found to be essential for transformation. Deletion of all or part of the region from amino acids 121 to 127 resulted in a total loss of transforming ability. An adjacent stretch of amino acids (residues 128 to 139), largely consisting of acidic residues, was found to be dispensable for transformation but appeared to influence the efficiency of transformation. Amino acids 1 to 85 made up a second region of the E1A protein that was essential for transformation. Deletion of all or part of this region resulted in a loss of the transforming activity. Even a mutation resulting in a single amino acid change at position 2 of the polypeptide chain was sufficient to eliminate transformation. Deletion of amino acids 86 to 120 or 128 to 289 did not eliminate transformation, although some mutations in these regions had lowered efficiencies of transformation. Foci induced by transformation-competent mutants could be expanded into cell lines that retained their transformed morphology and constitutively expressed the mutant E1A proteins.