Roles ofp53 and the E1B-55K protein in adenovirus replication: Implications for adenovirus-based oncolytic therapies

The Ad5 E1B-55K protein has been shown to impact many critical stages of the virus' life cycle. In the early phase of infection, E1B-55K counters the E1A-induced stabilization of p53 that may otherwise lead to the growth arrest or apoptotic death of the host cell, and in the late phase, E1B-55K cooperates with E4-orf6 to facilitate the efficient nucleo-cytoplasmic transport and translation of the late viral mRNAs. It was recently suggested that an E1B-55K-deleted adenovirus (dl1520) could preferentially replicate in p53-negative tumor cells, but not in p53-positive tumor cells where replication would be attenuated by p53. It was additionally proposed that this virus (renamed ONYX-015) might be used therapeutically as a “magic bullet” to selectively destroy cancerous cells while sparing the surrounding normal tissues.; These findings surprised us, as we could not understand how the absence of p53 within a host cell could abrogate the requirements for the p53-independent functions of E1B-55K in replication. We therefore reevaluated p53's role in determining the outcome of infection with an E1B-55K-deleted adenovirus. dl1520 replication was found to vary widely from cell line to cell line, and further did not correlate with the genotype or functional status of p53 in the cell. When p53's influence was examined away from the genetic background differences among the different cell lines however, p53 function was found to restrict viral DNA replication. The accumulation of late viral mRNAs in both the nucleus and cytoplasm were also affected. Moreover, we uncovered an additional late phase requirement for E1B-55K in ensuring the efficient translation of the late RNAs.; Despite the many processes in which E1B-55K has been implicated, the mechanism by which it performs its many functions is not well understood. The use of dl1520/ONYX-015 in the clinic, however, makes it important to understand the normal functions of the 55K protein. We thus undertook a proteomics-based approach to identify novel E1B-55K interacting proteins. A multi-protein, E1B-55K-containing complex was purified from Ad5-infected HeLa cells and found to contain E4-orf6, in addition to several cellular factors implicated in the ubiquitination of proteins. These factors included Cullin-5, Rbx1/Hrt1/ROC1, and the Elongins B and C. We demonstrate that these components exist in a single complex in vivo, and are further able to mediate p53 polyubiquitination in vitro. Our findings support a model in which E1B-55K functions as the principal substrate recognition component of an SCF-type ubiquitin ligase.