The E1A N terminus (aa 1-29) of the highly oncogenic adenovirus type 12 harbours a trans-activation function not detectable in the non-oncogenic serotype 2
Early region 1A (E1A) of adenoviruses (Ad) codes for potent activator and repressor molecules which are involved in the regulation of viral and cellular gene expression. Gene regulatory functions of E1A proteins are mainly located in their conserved regions (CR) 1 to 3. In addition to the CRs, specific amino acids (aa) of the N-terminal end play an important role in some gene regulatory functions. We describe here the identification and characterization of a novel trans-activation domain which is located in the non-conserved N-terminal end of Ad12 E1A, namely aa 1–29. Fusion of this region to the DNA-binding domain of the yeast transcription factor Gal4 generates a strong trans-activator which induces gene expression of reporter constructs in dependence on Gal4 DNA-binding sites. Furthermore, transient expression assays using the physiological E1A-responsive adenoviral E2 early promoter revealed that the N terminus is involved in its activation. The gene regulatory function of the N terminus is specific for E1A proteins of the highly oncogenic serotype Ad12, as the respective E1A N terminus of the non-oncogenic serotype Ad2 is unable to activate the expression of the reporter gene as Gal4 fusion protein. Moreover, deletion mutant analyses demonstrate that Ad12 E1A proteins carry three independently acting activation domains: (1) aa 1–29, (2) CR1 and (3) CR3.
AranyZ., NewsomeD., OldreadE., LivingstonD. M., EcknerR.1995; A family of transcriptional adaptor proteins targeted by the E1A oncoprotein. Nature 374:81–84
BondessonM., MannervikM., AkusjärviG., SvenssonC.1994; An adenovirus E1A transcriptional repressor domain functions as an activator when tethered to a promoter. Nucleic Acids Research 22:3053–3060
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
BrockmannD., EscheH.1995; Regulation ofviral and cellular gene expression by E1A proteins encoded by the oncogenic adenovirus type 12. Current Topics in Microbiology and Immunology 199/III:82–112
Byers KrausV., InostrozaJ. A., YeungK., ReinbergD., NevinsJ. R.1994; Interaction of the Dr1 inhibitory factor with the TATA binding protein is disrupted by adenovirus E1A. Proceedings of the National Academy of Sciences, USA 91:6279–6282
ByrdP. J., GrandR. J., GallimoreP. H.1988; Differential transformation of primary human embryo retinal cells by adenovirus E1 regions and combinations of E1A + ras. Oncogene 2:477–484
ChevrayP. M., NathansD.1992; Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proceedings of the National Academy of Sciences, USA 89:5789–5793
EganC., JelsmaT. N., HoweJ. A., BayleyS. T., FergusonB., BrantonP. E.1988; Mapping of cellular protein-binding sites on the products of early region 1A of human adenovirus type 5. Molecular and Cellular Biology 8:3955–3959
FergusonB., KripplB., AndrisaniO., JonesN., WestphalH., RosenbergM.1985; E1A 13S and 12S mRNA products made in Escherichia coli both function as nucleus-localized transcription activators but do not directly bind DNA. Molecular and Cellular Biology 5:2653–2661
GedrichR. W., BayleyS. T., EngelD. A.1992; Induction of AP-1 DNA-binding activity and c-fos mRNA by the adenovirus 243R E1A protein and cyclic AMP requires domains necessary for transformation. Journal of Virology 66:5849–5859
GormanC., MerlinoG., WillinghamM., PastanI., HowardB.1982; The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eucaryotic cells by DNA-mediated transfection. Proceedings of the NationalAcademy ofSciences, USA 79:6777–6781
HarlowE., WhyteP., FranzaB. R.JrSchleyC.1986; Association of adenovirus early-region 1A proteins with cellular polypeptides. Molecular and Cellular Biology 6:1579–1589
HouwelingA., van den ElsenP. J., van der EbA. J.1980; Partial transformation of primary rat cells by the leftmost 4·5 % fragment of adenovirus 5 DNA. Virology 105:537–550
InostrozaJ. A., MermelsteinF. H., HaI., LaneW. S., ReinbergD.1992; Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription. Cell 70:477–489
JelinekT., PereiraD. S., GrahamF. L.1994; Tumorigenicity of adenovirus-transformed rodent cells is influenced by at least two regions of adenovirus type 12 early region 1A. Journal of Virology 68:888–896
JochemsenH., DaniëlsG. S. G., HertoghsJ. J. L., SchrierP. I., van den ElsenP. J., van der EbA. J.1982; Identification of adenovirus- type 12 gene products involved in transformation and oncogenesis. Virology 122:15–28
JonesN., ShenkT.1979; An adenovirus type 5 early gene function regulates expression of other early genes. Proceedings of the National Academy of Sciences, USA 76:3665–3669
LiuF., GreenM. R.1990; A specific member of the ATF transcription factor family can mediate transcription activation by the adenovirus E1A protein. Cell 61:1217–1224
LundbladJ. R., KwokR. P. S., LauranceM. E., HarterM. L., GoodmanR. H.1995; Adenoviral E1A-associated protein p300 as a functional homologue of the transcriptional co-activator CBP. Nature 374:85–88
MillerM. E., EngelD. A., SmithM. M.1995; Cyclic is required for function of the N-terminal and CR1 domains of adenovirus E1A in Saccharomyces cerevisiae. Oncogene 11:1623–1630
MurphyM., OpalkaB., SajaczkowskiR., Schulte-HolthausenH.1987; Definition of a region required for transformation in E1A of adenovirus 12. Virology 159:49–56
PereiraD. S., RosenthalK. L., GrahamF. L.1995; Identification of adenovirus E1A regions which affect MHC class I expression and susceptibility to cytotoxic T lymphocytes. Virology 211:268–277
SimonM. C., FischT. M., BeneckeB. J., NevinsJ. B., HeintzN.1988; Identification of multiple, functionally distinct TATA elements,one of which is the target in the hsp70 promoter for E1A regulation. Cell 52:723–729
WangH. -G. H., RikitakeY., CarterM. C., YaciukP., AbrahamS. E., ZerlerB., MoranE.1993a; Identification of specific adenovirus E1A N-terminal residues critical to the binding of cellular proteins and to the control of cell growth. Journal of Virology 67:476–488
WangH. -G. H., YaciukP., RicciardiR. P., GreenM., YokoyamaK., MoranE.1993b; The E1A products of oncogenic adenovirus serotype 12 include amino-terminally modified forms able to bind the retinoblastoma protein but not p300. Journal of Virology 67:4804–4813
WilliamsJ., WilliamsM., LiuC., TellingG.1995; Assessing the role of E1A in the differential oncogenicity of group A and group C human adenoviruses. Current Topics in Microbiology and Immunology 199/III:149–175
WuL., RosserD. S. E., SchmidtM. C., BerkA.1987; A TATA box implicated in E1A transcriptional activation of a simple adenovirus 2 promoter. Nature 326:512–515
The E1A N terminus (aa 1-29) of the highly oncogenic adenovirus type 12 harbours a trans-activation function not detectable in the non-oncogenic serotype 2