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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

Recent studies have indicated that a number of long noncoding RNAs (lncRNAs) are dysregulated in hepatocellular carcinoma, while their aberrant expressions are associated with tumorigenesis and poor prognosis. To identify hepatitis B virus (HBV)-related lncRNAs, we used RNA deep sequencing to quantify the abundances of lncRNAs in HepG2 cells and HBV transgenic HepG2-4D14 cells. Here, we demonstrate that lnc-HUR1 is significantly upregulated in HepG2-4D14 cells. We found that HBV-encoded hepatitis B x protein can enhance the transcription of lnc-HUR1. Overexpression of lnc-HUR1 promotes cell proliferation, whereas knockdown of lnc-HUR1 inhibits cell growth. We identified that lnc-HUR1 can interact with p53 and inhibit its transcriptional regulation on downstream genes, such as p21 and B cell lymphoma 2-associated X protein. We generated lnc-HUR1 transgenic mice and performed the partial hepatectomy (PHx) to examine liver regeneration. The data showed that the ratio of liver weight to body weight in lnc-HUR1 transgenic mice is higher than that in wild-type (WT) littermates at day 2 and day 3 following hepatectomy. Consistently, the results of bromodeoxyuridine staining on liver sections following hepatectomy indicate that the ratio of bromodeoxyuridine-positive cells in lnc-HUR1 transgenic mice is significantly higher than that in WT mice, suggesting that lnc-HUR1 promotes cell proliferation during liver regeneration. Next, we performed the experiment of diethylnitrosamine-induced tumorigenesis. The data demonstrate that tumor number in lnc-HUR1 transgenic mice is higher compared with control mice, indicating that lnc-HUR1 enhances diethylnitrosamine-induced tumorigenesis. Conclusion: We reveal that HBV-upregulated lnc-HUR1 promotes cell proliferation and tumorigenesis by interacting with p53 to block downstream gene transcription. Our findings suggest that lnc-HUR1 plays an important role in HBV-related hepatocellular carcinoma development and may serve as a therapeutic marker for hepatocellular carcinoma. (Hepatology 2018; 00:000-000).

Post-translational modifications of viral proteins play important roles in regulating viral replication. Here we demonstrated that the PB2 of influenza A virus (IAV) can be modified by NEDD8. We revealed that E3 ligase HDM2 can promote PB2 NEDDylation. Overexpression of either NEDD8 or HDM2 can inhibit IAV replication, while knockdown of HDM2 has the opposite effect. Then we identified residue K699 in PB2 as the major NEDDylation site. We found that NEDDylation deficient PB2 mutant (PB2 K699R) has a longer half-life than wild-type PB2, indicating that NEDDylation of PB2 reduces its stability. We generated an IAV mutant in which PB2 was mutated to PB2 K699R (WSN-PB2 K699R) and examined the replication of WSN and WSN-PB2 K699R viruses in both MDCK and A549 cells and found that the replication of WSN-PB2 K699R was more efficient than wild-type WSN. In addition, we observed that overexpression of NEDD8 significantly inhibited the replication of WSN, but not WSN-PB2 K699R. The infection assay in mice showed that WSN-PB2 K699R exhibited enhanced virulence in mice compared to WSN, suggesting that NEDDylation of PB2 reduced IAV replication in vivo. In conclusion, we demonstrated that NEDDylation of PB2 by HDM2 negatively regulates IAV infection.