Hepatitis B virus X protein induces E6-associated protein-mediated proteasomal degradation of p53 phosphorylated at Ser-15.

Abstract

The hepatitis B virus (HBV) X protein (HBx) activates the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) pathway, leading to phosphorylation of p53 at multiple sites including Ser-15. This phosphorylation largely contributes to p53 accumulation by preventing its degradation via MDM2. This study further shows that HBx facilitates E6-associated protein (E6AP)-mediated proteasomal degradation of p53 during HBV replication. In the presence of HBx, E6AP expression induced p53 ubiquitination, reduced its stability and decreased p53 levels, whereas E6AP knockdown elevated p53 levels. The critical role of E3 ligase activity in E6AP was confirmed using the E6AP (C833A) mutant, the homologous to the E6-AP Carboxyl Terminus (HECT)-type ubiquitin E3 ligase inhibitor Heclin and the proteasome inhibitor MG132. E6AP-mediated p53 degradation was severely impaired in the presence of the ATM inhibitor KU-55933, indicating that HBx-induced p53 phosphorylation plays a critical role in this process. Additionally, E6AP could target p53 phosphorylated by DNA-damaging agents like etoposide in the absence of HBx. HBx also strengthened the interaction between phosphorylated p53 and E6AP, suggesting an additional mechanism that enhances E6AP-mediated ubiquitination of phosphorylated p53. Ser-15 phosphorylation was pivotal, as E6AP could degrade p53 S15D (phosphomimetic mutant) but failed to act on p53 S15A (a non-phosphorylatable mutant). These findings suggest that HBx-induced p53 phosphorylation enhances E6AP-mediated degradation while concurrently inhibiting MDM2-mediated pathways, thereby fine-tuning p53 levels to support cell survival, viral replication and potentially carcinogenesis during HBV infection in human hepatocytes.