HDM2 promotes NEDDylation of HBV HBx to enhance its stability and function.

Abstract Hepatitis B virus-encoded X protein (HBx) plays a critical role in HBV-related hepatocarcinoma development. In this study, we demonstrated that HBx is specifically modified by NEDD8. We found that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability by preventing its ubiquitination-mediated degradation. Consistently, analysis of 160 HCC patient specimens indicated that the amount of HDM2 protein correlates with HBx protein level. We identified that HBx K91 and K95 as the key HBx NEDDylation sites and observed that the NEDDylation-deficient HBx has shorter half-life. We generated Huh7 cell lines which ectopically express wild-type and NEDDylation deficient HBx, and found that NEDDylation-deficient HBx showed less chromatin localization and less DDB1 binding. Consistently, the expression of HBx-regulated genes (IL-8, MMP9 and YAP) and the HBV transcription (the activity of HBV enhancer and the amount of pgRNA transcribed from cccDNA) were significantly higher in cells expressing WT HBx than that in cells expressing mutant HBx. In addition, HBx-expressing cells proliferated faster than control and mutant HBx-expressing cells. We also showed that the ability of WT HBx-expressing cells to form tumor in nude mice was significantly higher than that of mutant HBx-expressing cells. In conclusion, we revealed that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability and chromatin localization which in turn favors HBx-dependent transcriptional regulation, cell proliferation and promoting HBV-driven tumor growth.IMPORTANCE HBV HBx protein plays a critical role in viral replication and hepatocarcinogenesis. However, the regulation of HBx stability is not well understood. We found that HBx is modified by NEDD8 and that the HDM2 E3 ligase promotes HBx NEDDylation to enhance HBx stability by inhibiting its ubiquitination. We provide a new evidence to show the positive correlation between HDM2 and HBx in clinical HCC samples. We also identified the major Neddylation sites on HBx. Our studies indicate that the defective NEDDylation of HBx negatively affects its ability to activate transcription of downstream genes, promote cell proliferation and tumor growth in vivo Taken together, we revealed a novel posttranslational modification of HBx by HDM2 which regulates its stability, subcellular localization and functions. These findings indicate that HDM2 is an important regulator on HBx and a potential diagnosis/therapeutic marker for HBV-associated HCC.