p53 activity during adenovirus infection

Adenovirus is a small DNA tumour virus that has been extensively studied, leading to fundamental discoveries in molecular biology of mammalian cells. In particular, adenovirus oncoproteins were shown to inactivate cellular tumour suppressor pathways and this advanced our understanding of molecular mechanisms of cancer formation. p53 is a key tumour suppressor that ensures cellular genomic integrity. Its function is impaired in most human malignancies, and DNA tumour viruses evolved multiple ways to block p53 activity in favour of productive virus infection. Human adenovirus type 5 codes for two oncoproteins, E1B-55 kDa and E4-34 kDa that bind and forward p53 to degradation in proteasomes. Deletion or mutation of E1B-55 kDa leads to a massive accumulation of the p53 protein in infected cells. Based on this fact, an idea of p53-selective replicating oncolytic virus for cancer treatment was proposed. It was assumed that an infection of cancer cells containing mutant or null p53 with E1B-deficient virus should allow unrestricted virus replication and cell lysis. In contrast, normal cells, bearing wild type p53, were expected to block the replication of such virus mutants, since functional p53 should induce cell cycle arrest or apoptosis after infection. This attractive idea was implemented by the creation of an E1B-55 kDa-deleted oncolytic virus, designated ONYX-015. However, in spite of moderate successes in head and neck cancer treatment, ONYX-015 did not become a breakthrough in tumour therapy. As was shown by numerous studies later, E1B-deficient virus replication was independent of the cellular p53 status, though indeed, the virus replicated better in some cancer cells as compared to normal cells. The reason for ONYX-015 failure was understood on the molecular level, when p53 activity was carefully examined after infection. It became clear that, despite massive accumulation of p53, E1B-deficient virus blocked its transcriptional activity and thus prevented cell cycle arrest or apoptosis induction in the infected cells. Therefore, we speculated that, in addition to E1B-55 kDa, adenovirus evolved back up mechanisms of p53 inactivation that were investigated in this study. First of all, we show here that infection with E1B-defective adenovirus mutants induces massive accumulation of p53, without obvious defects in p53 localization, phosphorylation, conformation, and oligomerization. Nonetheless, p53 completely failed to induce its target genes, e. g. p21/CDKN1A, mdm2, and PUMA. Secondly, we found the adenovirus E1A proteins to be responsible for blocking p53 activity in the absence of E1B-55 kDa. Two regions of the E1A gene products independently contributed to p53 suppression. Depending on the E1A conserved region 3 (CR3), E1B-defective virus blocked transcription of p53 target genes, and impaired the ability of the transcription factor Sp1 to bind the p21 promoter. Moreover, the aminoterminal region of E1A, binding the acetyltransferases p300 and CBP, blocked p53 K382 acetylation in infected cells. Mutating either of these E1A regions, in addition to E1B, partially restored p53 activity. We conclude that adenovirus inactivates p53 by at least two E1B-independent mechanisms. Thus, our study provides a mechanistic explanation why the lack of the E1B-55 kDa cannot be expected to result in p53-selective cytotoxicity. The mechanisms of p53 inactivation by E1A, described here, should be taken into account, when attempting to create p53-selective adenovirus for cancer therapy. Our findings may also help to understand the molecular mechanisms of p53 attenuation by other virus species and by virus-independent cancer cells.