Database of mutations within the adenovirus 5 E1A oncogene.

The Ad5 E1A database is a listing of mutations affecting the early region 1A (E1A) proteins of human adenovirus type 5. The database contains the name of the mutation, the nucleic acid sequence changes, the resulting alterations in amino acid sequence and reference. Additional notes and references are provided on the effect of each mutation on E1A function. The database is contained within the Adenovirus 5 E1A page on the World Wide Web at: http://www.geocities.com/CapeCanaveral/Hangar /2541/     

Interactions between cell growth-regulating domains in the products of the adenovirus E1A oncogene.

Among the various biological activities expressed by the products of the adenovirus E1A gene are the abilities to induce cellular DNA synthesis and proliferation in quiescent primary baby rat kidney cells. The functional sites for these activities lie principally within two regions of the E1A proteins: an N-terminal region and a small second region of approximately 20 amino acids further downstream. To study the biological functions of the first domain, we constructed an in-frame deletion of amino acid positions 23 through 107 of the E1A products. This deletion did not impede the ability of the E1A products to transactivate the adenovirus early region 3 promoter in a transient-expression assay in HeLa cells. The ability to induce DNA synthesis in quiescent baby rat kidney cells was, however, lost in the absence of these sequences. Deletion of the small second region induced a form of S phase in which DNA synthesis occurred in the apparent absence of controls required for the cessation of DNA synthesis and progression through the remainder of the cell cycle. These cells did not appear to accumulate in or before G2, and many appeared to have a DNA content greater than that in G2. The functions of both domains are required for production of transformed foci in a ras cooperation assay. Focus formation occurred, however, even when the two domains were introduced on two separate plasmids. This complementation effect appeared to require expression of both of the mutant proteins and did not appear to result merely from recombination at the DNA level.     

Adenovirus type 5 E1A gene products act as transformation suppressors of the neu oncogene.

The adenovirus type 5 early region 1A (E1A) gene was introduced into neu-transformed B104-1-1 cells. Cells that expressed E1A possessed reduced transforming activity in vitro and reduced tumorigenicity in nude mice. These results demonstrate that the E1A gene products can act negatively to suppress the transformed phenotype in neu-transformed cells.     

Mapping of cellular protein-binding sites on the products of early-region 1A of human adenovirus type 5.

The binding sites for the 300-, 107-, and 105-kilodalton cellular proteins which associate with human adenovirus type 5 E1A products were studied with E1A deletion mutants. All appeared to bind to the amino-terminal half of E1A products in regions necessary for oncogenic transformation. These results suggest that these cellular species may be important for the biological activity of E1A products.     

Cis effect of the type 5 adenovirus E1A gene enhancer element on cellular transformation

Mutants of type 5 adenovirus that lack all or part of the early region 1A (E1A) gene enhancer element transform rodent embryo fibroblast (CREF) cells at higher efficiencies than wild-type virus. An analysis of viral E1A cytoplasmic mRNA levels in mutant and wild-type virus-infected CREF cells revealed no differences in the levels of the E1A mRNAs. This implies that a decrease in the rate of viral E1A gene expression was not responsible for the transforming properties of the enhancer-less viruses. Unlike wild-type virus, however, the mutant viruses were able to replicate their genomes in the normally nonpermissive CREF cells. This change in viral DNA template concentration further resulted in an increase in early gene mRNA concentrations in mutant-virus-infected CREF cells. These studies suggest several possible mechanisms that could be responsible for the increased transforming potentials of these viruses, including 1) a cis effect of removing the viral E1A enhancer element on the efficiency of viral DNA integration, 2) viral DNA replication, or 3) an increase in the levels of the viral E1A and E1B mRNAs owing to viral DNA replication in the virus-infected CREF cells.     

Quantitative analysis of regions of adenovirus E1A products involved in interactions with cellular proteins.

Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36-69 within conserved region 1 (CR1), and residues 121-138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably.     

Constructing chimeric type 12/type 5 adenovirus E1A genes and using them to identify an oncogenic determinant of adenovirus type 12.

The E1A gene of highly oncogenic type 12 adenovirus (Ad12) possesses a segment unique to this serotype and comprising 60 base pairs contiguous with and separating conserved regions 2 and 3 in the gene. A similar but slightly longer segment is also present in the E1A gene of highly oncogenic simian adenovirus type 7 (D. Kimelman, J. S. Miller, D. Porter, and B. E. Roberts, J. Virol. 53:399-409, 1985). This segment is missing entirely from the E1A gene of type 5 adenovirus, which is nononcogenic. To test the hypothesis that this unique separating or "spacer" region influences the oncogenicity of Ad12, we constructed ClaI and SmaI restriction sites on either side of it, which allowed reciprocal exchange between this and the equivalent cassette from type 5 adenovirus E1A, bounded by the same restriction sites intrinsic to that gene. The resultant Ad12-based chimeric viruses, ch702 and ch704, in which the spacer region is replaced with (in-frame) type 5 sequence, grow normally on human A549 cells and display wild-type transformation frequencies on baby rat and mouse kidney cells. In contrast, the oncogenic capacity of these chimeric viruses, as measured by tumor induction following virus inoculation in Hooded Lister rats, is greatly reduced. Likewise, cells transformed by ch702 and ch704 display reduced tumorigenicity compared with wild-type transformants in syngeneic rats. These results, coupled with recent preliminary tests using a mutant with a point mutation in this region, support the view that the unique spacer region of type 12 is an oncogenic determinant of this virus.     

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.     

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.     

Alterations in cellular phenotype induced by the type 5 adenovirus E1A and the beta 1 protein kinase C genes in cloned rat embryo fibroblast cells.

The E1A gene of adenovirus type 5 (Ad5) induces morphological transformation and anchorage-independent growth in cloned rat embryo fibroblast (CREF) cells. In contrast, CREF cells transfected with a beta 1 protein kinase C (PKC) gene and expressing low-levels of beta 1 PKC display a CREF-like morphology and do not form colonies when grown in agar. The combination of Ad5 E1A and low-level beta 1 PKC expression in the same CREF cell line results in an enhanced ability to grow when suspended in agar. In Ad5 E1A and Ad5 E1A + low-level beta 1 PKC expressing CREF clones, the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further enhances anchorage-independence. In contrast, TPA does not induce CREF cells or transfected CREF cells expressing low-levels of beta 1 PKC to grow in agar. Low-level beta 1 PKC expression in transfected CREF cells is associated with a modest 1.2 to 1.6-fold increase in binding of [3H]-phorbol-12,13-dibutyrate (PDBu) and only a 2.3-fold increase in PKC enzymatic activity. In contrast, specific beta 1 PKC-retroviral vector transformed CREF clones (CREF-RV-PKC) display higher levels of PKC mRNA, PDBu binding and PKC enzymatic activity. A majority of CREF-RV-PKC clones exhibit a transformed morphology and grow more rapidly in monolayer culture, form macroscopic colonies in agar in the absence of TPA and in many independent clones TPA further enhances anchorage-independent growth. This effect is not directly related to the level of enhanced [3H]-PDBu binding. The present study indicates that the effect of beta 1 PKC on cellular phenotype in immortal rat embryo cells is complex and is affected by its mode of insertion into CREF cells, i.e. transfection versus retroviral insertion. In addition, the combination of a transfected Ad5 E1A and a beta 1 PKC gene in the same CREF clone results in an enhanced expression of the transformed phenotype in both the absence and presence of TPA.     

Different activities of the adenovirus types 5 and 12 E1A regions in transformation with the EJ Ha-ras oncogene.

We have compared the capacities of the E1A regions of nononcogenic adenovirus type 5 (Ad5) and highly oncogenic Ad12 to cooperate with the EJ bladder carcinoma Ha-ras-1 oncogene in the transformation of primary baby rat kidney cells. Both E1A regions, when cotransfected with the Ha-ras oncogene, transformed the primary cells with a low frequency. Ad5 E1A plus Ha-ras-transformed cells differed in phenotype from cells transformed by Ad12 E1A plus Ha-ras. The cells expressing Ad5 E1A appeared highly transformed and practically failed to adhere to plastic. This phenotype may be due to the virtually complete absence of fibronectin gene expression in these cells. In contrast, the cells expressing Ad12 E1A were flatter and adhered to plastic, whereas fibronectin gene expression was reduced but not absent. The oncogenic potential of the two types of E1A plus ras-transformed cells was tested by their injection into both athymic nude mice and weanling syngeneic rats. The Ad5 E1A plus ras-transformed cells were found to be highly oncogenic in both animal species, whereas the Ad12 E1A plus ras-transformed cells were only weakly oncogenic in both syngeneic rats and nude mice. The difference in oncogenic potential of the Ad5 E1A plus ras- and the Ad12 E1A plus ras-transformed cells is discussed in terms of the different capacities of the Ad5 and Ad12 E1A-encoded proteins to modulate cellular gene expression.     

Intranuclear location of the adenovirus type 5 E1B 55-kilodalton protein.

The intracellular location of the adenovirus type 5 E1B 55-kilodalton (kDa) protein, particularly the question of whether it is associated with nuclear pore complexes, was examined. Fractionation of adenovirus type 5-infected HeLa cell nuclei by an established procedure (N. Dwyer and G. Blobel, J. Cell. Biol. 70:581-591, 1976) yielded one population of E1B 55-kDa protein molecules released by digestion of nuclei with RNase A and a second population recovered in the pore complex-lamina fraction. Free and E1B 55-kDa protein-bound forms of the E4 34-kDa protein (P. Sarnow, C. A. Sullivan, and A. J. Levine, Virology 120:387-394, 1982) were largely recovered in the pore complex-lamina fraction. Nevertheless, the association of E1B 55-kDa protein molecules with this nuclear envelope fraction did not depend on interaction of the E1B 55-kDa protein with the E4 34-kDa protein. Comparison of the immunofluorescence patterns observed with antibodies recognizing the E1B 55-kDa protein or cellular pore complex proteins and of the behavior of these viral and cellular proteins during in situ fractionation suggests that the E1B 55-kDa protein does not become intimately or stably associated with pore complexes in adenovirus-infected cells.