Hepatitis B virus (HBV)-encoded X protein (HBx) plays a critical role in HBV-related hepatocarcinoma development. In this study, we demonstrate that HBx is specifically modified by NEDD8. We found that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability by preventing its ubiquitinationmediated degradation. Consistently, analysis of 160 hepatocellular carcinoma 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 HBV transcription (the activity of HBV enhancer and the amount of pgRNA transcribed from cccDNA) were significantly higher in cells expressing wild-type (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 tumors 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 HBV-driven tumor growth. IMPORTANCE Hepatitis B virus (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 hepatocellular carcinoma (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 the transcription of downstream genes and promote cell proliferation and tumor growth in vivo. Taken together, our findings reveal 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 HBVassociated HCC.KEYWORDS HBx, NEDDylation, stability, chromatin localization, hepatocellular carcinoma, HDM2, hepatitis B virus
Hepatitis B virus (HBV) causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV persistence are not well understood. In this study, we found that HBV inhibited the chemotherapy drug etoposide-induced apoptosis of hepatoma cells. Further analysis revealed that HBV mRNAs possess a microRNA 15a/16 (miR-15a/16)-complementary site (HBV nucleotides [nt] 1362 to 1383) that acts as a sponge to bind and sequester endogenous miR-15a/16. Consequently, Bcl-2, known as the target of miR-15a/16, was upregulated in HBV-infected cells. The data from HBV-transgenic mice further confirmed that HBV transcripts cause the reduction of miR15a/16 and increase of Bcl-2. More importantly, we examined the levels of HBV transcripts and miR-15a/16 in HBV-infected HCC from patients and found that the amount of HBV mRNA and the level of miR-15a/16 were negatively correlated. Consistently, the level of Bcl-2 mRNA was upregulated in HBV-infected patients. In conclusion, we identified a novel HBV mRNAmiR-15a/16 -Bcl-2 regulatory pathway that is involved in inhibiting etoposide-induced apoptosis of hepatoma cells, which may contribute to facilitating chronic HBV infection and hepatoma development. There are approximately 350 million chronic hepatitis B virus (HBV) carriers worldwide, and chronic HBV infection is the major etiological factor in hepatocellular carcinoma (HCC) (1, 2). The relative risk for the development of HCC in chronic hepatitis B (CHB) patients is estimated to be 25 to 37 times higher than that in those without infection (1,3,4).HBV is an enveloped, partially double-stranded DNA virus with a genome size of 3.2 kb. The HBV genome contains four overlapping open reading frames (ORFs). The RNA transcripts are polyadenylated and capped and are named the pre-C/C or pregenomic RNA (pgRNA) and the pre-S, S, and X mRNAs. These mRNAs encode several viral proteins, including the polymerase, core, HBe, pre-S1, S2, S, and X proteins (5). HBV has been reported to play an important role in regulating apoptosis. For example, HBV core protein inhibits TRAIL-induced apoptosis of hepatocytes by blocking DR5 expression (6). HBx can bind to the C terminus of p53 and inhibit p53-mediated cellular processes, including transcriptional transactivation and apoptosis (7). But the HBx protein was also found to sensitize cells to apoptotic killing by tumor necrosis factor alpha (8) and to inhibit Fas-mediated apoptosis associated with upregulation of the SAPK/JNK pathway in Chang cells (9).MicroRNAs (miRNAs) are single-stranded noncoding RNAs which negatively regulate gene expression at the posttranscriptional level, primarily through base pairing to the 3=-untranslated region (UTR) of target mRNA (10). Growing evidence indicates that microRNAs control basic cell functions, ranging from proliferation to apoptosis, by direct targeting (11,12). For instance, miR-101 exerts a proapoptotic function by targeting Mcl-1 (13), and miR-29c inhibits ce...
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