Modulation of p53 function by adenovirus E1B58kDa

From many lines of evidence, there is now no doubt that the p53 gene encodes a protein which can act as a tumour suppressor. In a wide range of human malignancies the p53 tumour suppressor gene has been shown to be mutated which often leads to the inactivation of its multiple wild type activities. Several "hot spots" for mutation of p53 have been identified in tumours from specific tissues, suggesting their importance in the progression towards malignancy, and indeed there is direct evidence that mutant p53 enhances transformation. Mutations in the p53 gene are presently recognized as the most frequent genetic alteration in human cancers. One of the most studied characteristics of p53 is its ability to associate with a great variety of both viral and cellular proteins. These associations might provide another mechanism by which wild type p53 activities can be altered. The viral proteins that bind to p53 appear to disrupt its functioning to allow for efficient viral replication and transformation. One such viral protein is the ElB58kDa protein of_adenovirus which was studied in this thesis with respect to its modulation of p53 function. The aim of this thesis was to explore the biochemical and biological characteristics of the p53/ElB58kDa interaction. To this end, conditions for complex formation were assessed in Chapter 3. The p53/ElB58kDa complex was detected in virally transformed and adenovirus infected cells. In addition, the first evidence for the formation of this complex in vitro illustrated that other cellular or viral components were not required for the association between p53 and ElB58kDa. The amino terminal region of p53 was demonstrated to be necessary for binding to ElB58kDa and highly conserved between species. This amino terminal region undergoes temperature dependent conformational changes in Xenopus laevis which affect the binding of ElB58kDa to p53 and thereby its activity, as discussed in Chapter 4. In addition, another protein of approximately 160kDa also associated with the p53/ElB58kDa complex. This 160kDa protein is probably cellular in origin and bound to ElB58kDa in particular, however, its involvement in the activity of the p53/ElB58kDa complex remains to be elucidated. Once the biochemical characteristics of the p53/ElB58kDa complex were established, the ability of ElB58kDa to modulate several biological activities of p53 was investigated in vivo and is presented in Chapters 5 and 6. Although the ElB58kDa protein had no effect on transcriptional . Vt.t repression or induction of apoptosis by p53 in transient assays, it clearly influenced transactivation of gene expression. Transactivation mediated by p53 was either abrogated or enhanced depending upon the gene promoter examined. Modulation of transactivation by ElB58kDa provides a potent mechanism for the regulation of p53 targeted cell cycle inhibitory genes. This alteration of transactivation has the potential to effectively change the activity of p53 from a tumour suppressor to a dominant oncogene. In Chapter 6, examination of endogenous human p53 under conditions of viral infection provided another in vivo system to study the effects of ElB58kDa on the biological activity of p53. Human p53 was shown to be specifically shut-off in adenovirus infected cells. Complex formation was required for the shut-off of human p53 during adenovirus infection which appeared to occur by ElB58kDa repression of transactivation. The ability of ElB58kDa to shut-off p53 during infection appeared to be necessary for a productive adenovirus infection to proceed. In summary, the results from my studies have indicated that ElB58kDa can modulate some biological activities of wild type p53, but which ones depends on the biological system being studied. For example, in transient reporter assays, only p53 dependent gene activation is affected. However, in virus infected human cells expressing a wild type p53, presumably all p53 dependent activities are affected, as p53 synthesis is completely shut-off.