The ElA proteins of adenovirus, large T antigens of polyomaviruses such as SV40, and E7 proteins of human papillomavirus-16 have potent activities in standard transformation assays. Genetic and biochemical studies have shown that the transforming domains of each viral oncoprotein form stable complexes with cellular proteins, demonstrating a tight correlation between complex formation and transformation activity. Two of the ElA-associated proteins have been identified as the retinoblastoma tumor suppressor gene product and human cyclin A. Both of these proteins are known to have important roles in the control of cell cycle and proliferation, reinforcing the belief that ElA has targeted key regulatory proteins. We propose to investigate the cellular p3OO protein that is targeted by adenovirus ElA. Mutagenesis of EIA indicates that binding to p3OO is essential for efficient transformation, but the interaction with p3OO is genetically and physically distinct from ElA's association with the retinoblastoma protein and human cyclin A. Although p300 has many interesting properties, it has proven to be difficult to study. p300 is expressed at low levels and is known only through its association with ELA- We propose to take two approaches to investigate p300. Initially we will further characterize the ElA-p300 interaction. These data will be used correlate the exact binding sites of p300 with the regions needed for the various ElA-mediated activities that have been mapped to this region of ElA. In addition, we will construct a peptide able to bind specifically to p300 that will be used in a second group of experiments designed to purify p300 and clone its cDNA. Although we have developed one assay to monitor the purification of p300, the availability of the p300 binding peptide may be helpful in the development of an improved and more sensitive assay to follow p300 purification profiles. This reagent may also be useful in affinity purification schemes. The major difficulty in developing p300 purification has been the low levels of this protein in tissue culture cells. In the light of recent discoveries that p300 is conserved in many species and is widely expressed in different cells, we will be able to use animal tissues as a high volume source for p300. The goal of this second set of experiments will be to use the cloned cDNA for p300 as a tool in the further investigation of the p300 protein.
| Dyson, N; Guida, P; Munger, K et al. (1992) Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins. J Virol 66:6893-902 |
