The Regulation Of CUL4B On HBV Replication And Its Molecular Mechanisms

The hepatitis B virus (HBV) belongs to the family of hepatotropic DNA virus, which can cause an acute, chronic or fulminant hepatitis by specifically infecting hepatocytes. Although the availability of HBV vaccines leads to the decline of HBV infection rate, there are still2billion people infected with HBV worldwide. Approximately350million people are chronically infected with HBV. It has been confirmed that persistent HBV infection is a major risk factor for the development of hepatocellular carcinoma (HCC), which is dangerous to human healthy. Therefore, the study on the regulatory mechanisms of HBV replication is necessary and significant to find a potent target for the clearance of HBV infection and the blockade of the development of HBV related diseases.The ubiquitin proteasome system (UPS) pathway has been involved in regulating a variety of biological processes by mediating the protein ubiquitination and promoting the proteasomal degradation, and thus plays an important role in maintaining the homeostasis of the cell and human body. In eukaryotic cells, ubiquitin activating E1, ubiquitin conjugating E2and ubiquitin ligase E3together catalyze the transfer reaction of ubiquitin molecules and promote the degradation of ubiquitinated proteins. Moreover, E3could specially recognize the substrate proteins and mediate the combination of ubiquitins with indicated proteins. Therefore, E3ligase exerts an important role in the UPS pathway. In recent years, the UPS pathway has been identified to be involved in the regulation of HBV replication cycle. The inhibition of proteasome with bortezomib markedly reduced the level of HBV DNA in HBV-transgenic mice. Meanwhile, Inhibition of HBV replication by Interferon requires proteasome activity. However, another group found that the proteasome inhibitor MLN-273could promote HBV replication in HBV-transgenic mice and C57BL/6mice injected with HBV whole genome plasmids. Therefore, the exact role of UPS pathway on HBV replication remains unclear.CUL4B, one member of the Cullin-RING gene family, has been identified as a novel candidate gene for X-linked mental retardation disorders. The mutation of this gene could lead to mental retardation, aphasia, huge tongue, short stature et al. Up to now, eight Cullin family genes have been identified including CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, CUL7, and PARC. And each of them could assemble a distinct of E3complexes, which is involved in the regulation of cell cycle, cell proliferation and differentiation, DNA replication and repair, signal transduction, viral infections and other cellular processes of life. In conclusion, they play very important roles in maintaining normal cellular biological functions.However, it is still unknown whether E3ubiquitin ligase CUL4B regulates HBV replication through the ubiquitin proteasomal pathway. Therefore, the present work is aiming to explore the role of CUL4B in HBV infection and to study the underlying molecular mechanisms.Objectives:1. To study the effect of CUL4B on HBV replication.2. To explore the molecular mechanisms of CUL4B regulation on HBV replication,and to provide a scientific basis for antiviral treatment.Methods:1. The regulation of CUL4B on HBV replication1.1The correlation between CUL4B and HBV replication in clinical samples and HBV-transgenic mice 1.1.1The correlation between CUL4B and HBV replication in human liver tissuesForty-three of HBV-positive liver tissue samples were collected. Liver tissue RNA was extracted and, CUL4B mRNA and HBV pgRNA were detected by real-time PCR. Meanwhile, liver tissue genome DNA was extracted and HBV cccDNA was also detected by real-time PCR. The correlation between CUL4B expression and HBV replication was statistically analyzed.1.1.2The correlation between CUL4B and HBV replication in HBV-transgenic miceLiver tissue RNA from6-8weeks old, male, HBV Tg BalB/c mice was extracted and CUL4B mRNA and HBV pgRNA were detected by real-time PCR. Meanwhile, genome DNA was extracted and HBV cccDNA was also detected by real-time PCR. Additionally, HBV DNA in blood serum of HBV Tg mice was also detected by real-time PCR. The correlation between CUL4B expression and HBV replication was statistically analyzed.1.2The regulation of CUL4B on HBV replication in vivo1.2.1The effect of CUL4B on HBV replication in CUL4B transgenic mice6-8weeks old Cul4b transgenic mice (CD1background, EGFP-CUL4B Tg) and wild type mice were hydrodynamically injected with HBV genome expression plasmids by tail-vein injection. The levels of HBsAg and HBeAg in blood serum were then detected by ELISA. And the expression of CUL4B mRNA, HBV cccDNA and HBV pgRNA in liver tissue was detected by real-time PCR. The correlation between CUL4B expression and HBV replication was statistically analyzed.1.2.2The effect of CUL4B on HBV replication in Mxl-Cre CUL4BFlox/Y conditional knock-out mice6-8weeks old, male, conditional CUL4B knock-out mice (C57BL/6background, Mxl-Cre CUL4BFlox/Y) and wild type mice were treated by PIPC.5days later, these mice were hydrodynamically injected with pcDNA3-HBV1.1plasmids. The level of HBsAg and HBeAg in blood serum was detected by ELISA. The expression of CUL4B mRNA, HBV cccDNA and HBV pgRNA in mouse liver was analyzed by real-time PCR. The correlation between CUL4B expression and HBV replication was statistically analyzed.1.3Regulation of CUL4B on HBV replication in vitroHepG2.2.15cell was transfected with CUL4B-mi expression plasmids or negative control Neg-mi expression plasimds. BEL7402cells, HepG2cells were co-transfected with HBV whole genome expression plasmids together with CUL4B-miRNA or negative control Neg-miRNA plasmids. In addition, Flag-CUL4B or Flag-empty together with HBV whole genome expression plasmids were co-transfected into HepG2cells. The efficiency of CUL4B interference or overexpression was verified by Western blot and real-time PCR. The levels of HBsAg and HBeAg in the cell supernatants were detected by ELISA. The expression of HBV cccDNA and HBV pgRNA were determined by real-time PCR. The role of CUL4B over-expression or silence on HBV replication was then analyzed.2. The involvment of HBx in regulation of CUL4B on HBV replicationHepG2cells were co-transfected with CUL4B over-expression plasmids or control plasmids together with pcDNA3-HBV1.1(wild type group) or pcDNA3-HBV1.1(â–³HBx)(HBx-deficiency group) or pcDNA-HBV1.1(â–³HBx)+pcDNA3-HBx-HA (HBx rescue group). The levels of HBsAg and HBeAg in cell supernatants were detected by ELISA. The expression of CUL4B mRNA, HBV pgRNA and HBV cccDNA was detected by real-time PCR. The role of HBx in the CUL4B regulating HBV replication was analyzed.3. Roles of HBx protein in the regulation of CUL4B on HBV replication3.1HBx interacts with CUL4B ubiquitin ligase complexHEK293cells were transfected with HBx over-expression plasmids.24hours later, the whole cell lysates were harvested with lysing buffer for co-immunoprecipitation (IP) assay with HA antibody or CUL4B anbibody. Western blot was used to detect the expression of CUL4B, DDB1, Rocl and HBx-HA to verify the interaction between HBx protein and CUL4B complex.3.2The effect of CUL4B ubiquitin ligase complex on the expression of HBx HEK293cells, HepG2cells and SMMC7721cells were co-transfected with HBx expression plasmids and CUL4B over-expression plasmids or CUL4B-miRNA or control plasmids. The effect of CUL4B on the expression of HBx was verified by RT-PCR and Western blot. Moreover, HEK293and Hela cells were co-transfected with CUL4B rescue plasmids and HBx expression plasmids, and the expression of HBx proteins were detected by Western blot. In addition, HBx expression plasmids and DDB1siRNA or ROC1siRNA were co-transfected into HKE293cells, and HBx protein expression was detected by Western blot in order to verify the role of DDB1and ROC1on the expression of HBx protein.3.3The effect of CUL4B on the half-life of HBx proteinHBx over-expression plasmids were transfected into CUL4B knockdown HEK293cells or control HEK293cells. Protein synthesis inhibitor (cycloheximide, CHX) was used to treat HEK293cells, and then cell proteins were collected at different time points. The HBx protein expression was detected by western blot. The half-life of HBx protein were calculated relative to the0level.3.4The regulation of CUL4B on HBx protein degradationHela, HEK293, HepG2and SMMC7721cells were co-transfected with CUL4B siRNA or negative control NC siRNA and HBx expression plasmids, followed by treatment with MG132(proteasome inhibitor) and HBx protein expression was then detected by Western blot.3.5The role of CUL4B on HBx protein ubiquitinationHBx over-expression plasmids were transfected into CUL4B knockdown HEK293cells or control HEK293cells. Then cells were treated with MG132and detected with HA antibody for IP assay. The HBx protein ubiquitination level was detected by Western blot using ubiquitin antibody.Results1. CUL4B positively regulates HBV replication1.1CUL4B expression positively correlates with HBV replication in clinical samples and HBV Tg mice 1.1.1CUL4B expression positively correlates with HBV replication in human liver tissuesThe expression of CUL4B, HBVpgRNA and HBV cccDNA in HBV positive liver tissues was detected by real-time PCR and statistically analyzed. Results showed that CUL4B mRNA expression level positively correlates with HBV pgRNA and HBV cccDNA levels in the HBV positive liver tissues.1.1.2CUL4B expression positively correlates with HBV replication in HBV transgenic miceThe expression of CUL4B, HBV pgRNA and HBV cccDNA in HBV Tg mouse liver and HBV DNA in blood serum were detected by real-time PCR. Statistical data indicate that CUL4B mRNA expression level positively correlates with HBV pgRNA and HBV cccDNA level in liver and HBV DNA level in the blood serum from HBV Tg mouse.1.2CUL4B promotes HBV replication in vivo1.2.1CUL4B transgenic mice promotes HBV replicationCUL4B Tg mice and wild type controls were hydrodynamically injected with HBV expression plasmids. Data from real-time PCR showed that the expression level of CUL4B mRNA, pgRNA and cccDNA were greatly up-regulated in CUL4B Tg mice than that in control mice. ELISA showed that HBsAg and HBeAg levels in blood serum from CUL4B Tg mice were much higher than that from control mice. Above data indicate that high expression of CUL4B could positively regulate HBV replication.1.2.2Conditional CUL4B knockout mice inhibits HBV replicationMxl-Cre CUL4BFlox/Ymice and control mice were treated with PIPC and then hydrodynamically injected with HBV expression plasmids. Real-time PCR showed that CUL4B mRNA, pgRNA and cccDNA levels in Mxl-Cre CUL4BFlox/Ymice were significantly lower than that in control mice. ELISA data showed that the levels of HBsAg and HBeAg in blood serum from Mxl-Cre CUL4BFlox/Y mice were significantly lower than that in control mice. These data suggest that CUL4B could promote HBV replication in vivo. 1.3CUL4B positively regulates HBV replication in vitroCUL4B over-expression plasimids was transfected into liver cancer cell lines together with HBV expression plasimids. CUL4B overexpression upregulated the expression of pgRNA and cccDNA in cells and increased the HBsAg and HBeAg level in supernatants. On the contrary, pgRNA, cccDNA, HBsAg and HBeAg levels were downregulated when silencing CUL4B expression in liver cancer cell lines. These results verified that CUL4B could promote HBV replication in vitro.2HBx was involved in CUL4B regulating HBV replicationHepG2cells were co-transfected with CUL4B expression plasmids and HBV whole genome expression plasmids, HBx-deficient HBV plasmids (â–³HBx) orâ–³HBx plus HBx plasmids. Results show that the expression of pgRNA, cccDNA, HBsAg and HBeAg were significantly lower inâ–³HBx group than those in HBV whole genome expression plasmids group. Moreover, HBx expression plasmids could restore HBV replication inâ–³HBx group. CUL4B over-expression markedly up-regulated the expression of pgRNA, cccDNA, HBsAg and HBeAg in the cell transfected with HBV whole genome plasmids, but this role became weakened inâ–³HBx group. However, HBx over-expression could restore the promotion of CUL4B on HBV replication in HBx-decifient group.3CUL4B regulates the ubiquitynation and proteasomal degradation of HBx3.1HBx could combine with CUL4B ubiquitin ligase complexHEK293cells were transfected with HBx over-expression plasmids and harvested24h later with lysing buffer for IP assay. Data from Westren blot suggest that HBx could combine with CUL4B, DDB1and ROC1.3.2CUL4B ubiquitin ligase complex upregulates the expression of HBxLiver cancer cell lines, HEK293cells and Hela cells were transfected with CUL4B over-expression plasmids or interference plasmids. RT-PCR results showed that CUL4B over-expression or interference had no obvious roles on HBx mRNA expression. However, results from Western blot show that CUL4B over-expression up-regulated HBx protein expression, and CUL4B interference inhibited HBx protein expression. Moreover, CUL4B rescue plasmids restored the expression of HBx protein in low CUL4B-expressing cells in a dose-dependent way. In addition, HEK293cells were transfected with HBx plasmid together with DDB1siRNA or ROC1siRNA. Western blot showed that silence of DDB1or ROC1also downregulated the expression of HBx protein. These data suggest that CUL4B ubiquitin ligase complex could up-regulate HBx protein expression.3.3CUL4B prolongs the half-life of HBx proteinThe role of CUL4B on the half-life of HBx protein was evaluated for further studying the molecular mechanisms of CUL4B on HBx protein expression. CUL4B-knockdown HEK293cells were treated with CHX and harvested at different time points. Data showed that HBx protein expression was greatly reduced in CUL4B-knockdown HEK293cells compared with that in control cells. Moreover, the degradation rate of HBx protein was significantly higher in CUL4B-knockdown HEK293than that in control cells. Above data indicate that CUL4B may up-regulate HBx protein expression by prolonging the half-life of HBx protein3.4CUL4B inhibits the proteasomal degradation pathway of HBx proteinPrevious studies have proved that HBx protein is mainly degraded through UPS pathway. For studying the role of this pathway in the regulation of CUL4B on HBx expression, cells were treated with proteasomal inhibitor MG132. Western blot showed that HBx expression was markedly reduced when CUL4B was silenced, but MG132almost abrogated the inhibition of CUL4B knockdown on HBx protein expression. These results suggest that CUL4B may regulate HBx protein expression by UPS pathway3.5CUL4B inhibits the ubiquitination of HBx proteinThe role of CUL4B on HBx ubiquitination was detected for further studying the role of UPS pathway in the regulation of CUL4B on HBx protein expression. HA antibody was used for IP assay and ubiquitination level were detected by western blot with ubiquitin antibody. Results show that the ubiquitination level of HBx protein was significantly higher in CUL4B knockdown cells than that in control cells. These data indicate that CUL4B may block the UPS pathway of HBx protein through inhibiting HBx protein ubiquitination. Conclusions and significances:1This study firstly finds that CUL4B E3ubiquitin ligase promote HBV replication, which may provide important evidence for illustrating the role of CUL4B in human diseases especially in virus infection related diseases.2HBx protein has been verified to have important roles in CUL4B regulating HBV replication, which uncovered a new molecular mechanism for HBx protein being involved in HBV replication.3This study firstly demonstrates that CUL4B ubiquitin ligase complexs combine with HBx, inhibit the degradation of HBx protein and finally up-regulate HBx protein expression. These findings provide new evidence for illustrating the UPS pathway in regulating HBV replication.