| BackgroundHepatitis B infection is a major public health problem as chronic HBV infection predisposes patients to severe liver disease, including decompensated cirrhosis and hepatocellular carcinoma, which seriously threaten people’s health. There is a high incidence of Hepatitis B infection in China.Therefore, understanding the regulation on HBV replication is important for the control of HBV infection and ultimately its elimination. Cullin 4B (CUL4B) is a scaffold protein of the CUL4B-RING-E3 ligase complex that mediates the ubiquitination of multiple intracellular proteins. Depending on the degradation of substrate, it can play a variety of important roles in the body, such as:DNA replication and damage repair, chromatin remodeling, cell cycle regulation, embryonic development, hematopoiesis. Our previous work has demonstratesd that CUL4B E3 ubiquitin ligase can promote HBV replication, and this function is partly dependent on HBx. Whether other proteins of Hepatitis B virus are involved in CUL4B regulation of HBV replication and the regulatory mechanism of HBx in this process is still unclear.During the infection of HBV, the outcome and progression are determined by interactions between virus virions and host immune system. It is widely accepted that adaptive immune responses play major roles in the defense of HBV infection. As the main target organ of hepatitis B virus, and also an important immune organ, liver’s innate immune function has been receiving increased attention in recent years. Thus, we also studied the role of hepatocyte CUL4B in regulating the innate immune cells by establishing acute hepatitis B mouse model.Objectives:1. To study the role and mechanism of viral proteins encoded by HBV in the regulation of HBV replication by CUL4B.2. To explore the molecular mechanisms of CUL4B regulation on HBV replication, and to provide a scientific basis for antiviral treatment.Methods1. The regulation of CUL4B on HBV replication1.1 Role of Polymerase in the regulation of HBV replication by CUL4B1.1.1 Verify the role of Polymerase in HBV replicationConstruction of Polymerase deletion mutants plasmid(pcDNA3-ΔPol). HepG2 cells were transfected with pcDNA3-HBV1.1 (Polymerase protein wild type group) or pcDNA3-HBV1.1 (APol) (Polymerase protein expression deficiency group) or pcDNA-HBV1.1 (APol) and pcDNA3-Polymerase (Polymerase rescue group). The levels of HBsAg in cell supernatants were detected by ELISA. The expression of HBV pgRNA was detected by PCR. The role of Polymerase in the HBV replication was analyzed based on above data.1.1.2 Analyze the role of Polymerase in the regulation of CUL4B on HBV replicationHepG2 cells were co-transfected with CUL4B over-expression plasmids or control plasmids along with pcDNA3-HBV1.1 (Polymerase protein wild type group) or pcDNA3-HBV1.1 (APol) (Polymerase protein expression deficiency group) or pcDNA-HBV1.1 (APol) and pcDNA3-Polymerase (Polymerase rescue group). The levels of HBsAg in cell supernatants were detected by ELISA. The expression of HBV pgRNA was detected by PCR. The role of Polymerase in the CUL4B regulating HBV replication was analyzed based on above data.1.2 Role of HBc in the regulation of HBV replication by CUL4B1.2.1 Verify the role of HBc in HBV replicationConstruction of Polymerase deletion mutants plasmid(pcDNA3-ΔHBc). HepG2 cells were transfected with pcDNA3-HBV1.1 (HBc protein wild type group) or pcDNA3-HBV1.1 (AHBc) (HBc protein expression deficiency group) or pcDNA-HBV1.1 (AHBc) and pcDNA3-HBc (HBc rescue group). The levels of HBsAg in cell supernatants were detected by ELISA. The expression of HBV pgRNA was detected by PCR. The role of Polymerase in the HBV replication was analyzed based on above data.1.2.2 Analyze the role of HBc in the regulation of CUL4B on HBV replicationHepG2 cells were co-transfected with CUL4B over-expression plasmids or control plasmids along with pcDNA3-HBV1.1 (HBc protein wild type group) or pcDNA3-HBV1.1 (AHBc) (HBc protein expression deficiency group) or pcDNA-HBV1.1 (AHBc) and pcDNA3-HBc (HBc rescue group). The levels of HBsAg in cell supernatants were detected by ELISA. The expression of HBV pgRNA was detected by PCR. The role of HBc in the CUL4B regulating HBV replication was analyzed based on above data.1.3 The role of HBx in CUL4B regulating HBV replication1.3.1 Analyze the effect of HBx on the expression of CUL4B.HepG2 cells were transfected with pcDNA3-HBV1.1(HBV intact genome),HBx,HBc and preS2 Fragments over-expression plasmids. The expression of CUL4B mRNA was detected by PCR, and the level of CUL4B protein were determined by Western blot. The interaction between HBx protein and CUL4B was analyzed based on above data.1.3.2 Study the role of HBx on the regulation of HBV replication by CUL4B in vitroHepG2 cells were co-transfected with CUL4B siRNA or negative control NC siRNA along with pcDNA3-HBV1.1 (HBx protein wild type group) or pcDNA3-HBV1.1 (AHBx) (HBx protein expression deficiency group) or pcDNA-HBV 1.1 (AHBx) andpcDNA3-HBx-HA (HBx rescue group). The levels of HBsAg and HBeAg in cell supernatants were detected by ELISA. The expression of HBV pgRNA was detected by PCR. The role of HBx in the CUL4B regulating HBV replication was analyzed based on above data.1.3.3 Study the role of HBx in the regulation of HBV replication in CUL4B in vivo6-8 weeks old, male, liver-specific Cul4b knock-out mice (C57BL/6 background, Alb-Cre;CUL4BFlox/Y) were injected with pcDNA3-HBV1.1 (ΔHBx) (HBx protein expression deficiency group) and pcDNA3 or pcDNA-HBV1.1 (ΔHBx) and pcDNA3-HBx-HA (HBx rescue group). The levels of HBsAg in blood serum were then detected by ELISA. And the levels of HBV DNA in blood serum was detected by real-time PCR. The role of HBx in the Cul4b regulating HBV replication was analyzed based on above data.1.4 Study the effect of CUL4B on the protein modification of HBV cccDNA minichromosomeIt has been reported that CUL4B regulates the expression of host genes by regulating histone methylation and ubiquitin modification, which are involved in the tumorigenesis. HBV covalently closed circular DNA (cccDNA) is organized into minichromosomes by histone and non-histone proteins. Remodeling of minichromosomes is crucial for the regulation of HBV replication. Therefore,in this study,we investigated whether CUL4B can combined with HBV cccDNA, and whether it can affect the histone modification status of HBV cccDNA minichromosome.1.4.1 Relationship between CUL4B and HBV cccDNAHuH-7 cells were co-transfected with prcccDNA and pCMV-Cre plasmids.The chromatin immunoprecipitation (ChIP) method was used to verify whether CUL4B could combined with cccDNA.1.4.2 Study whether CUL4B can altered the acetylation or methylation status of histone bound with cccDNAHuH-7 cells were co-transfected with CUL4B siRNA or negative control NC siRNA along with prcccDNA and pCMV-Cre plasmids. ChIP-qPCR method was used to verify whether CUL4B can altered the acetylation or methylation status of histone bound with cccDNA2. Effect of hepatocyte CUL4B on immune microenvironment of liver2.1 Effect of hepatocyte CUL4B expression on the proportion of innate immune cells in the liver6-8 weeks old, male, liver-specific CuI4b knock-out mice (C57BL/6 background, Alb-Cre Cul4bFlox/Y) were injected with pcDNA3-HBV1.1(HBV intact genome). The levels of HBV DNA in blood serum 24h post-injection was detected by real-time PCR.2.2 Effect of CUL4B expression on NK cell activation in the liverTotal mononuclear cells (MNC) were isolated from liver by Percoll. Flow cytometry was used to detect the proportion of lymphocytes in mononuclear cells.Results1. Mechanisms of CUL4B regulation on HBV replication1.1 Polymerase are not involved in CUL4B regulation on the replication of HBVPolymerase deletion mutants plasmid(pcDNA3-ΔPol) were constructed and been transfected into HepG2.Results show that the deletion of Polymerase doesn’t affect the promoting effect of CUL4B on HBV pgRNA, HBsAg and other viral replication indicators. Suggesting that Polymerase-deficiency had no effect on CUL4B regulated HBV-replication1.2 HBc are not involved in CUL4B regulation on the replication of HBVHBc deletion mutants plasmid(pcDNA3-ΔHBc) were constructed and been transfected into HepG2.Results show that the deletion of HBc doesn’t affect the promoting effect of CUL4B on HBV pgRNA, HBsAg and other viral replication indicators. Suggesting that HBc deficiency had no effect on CUL4B regulated HBV-replication1.3.1 HBx can promote the expression of CUL4BHepG2 cells were transfected with pcDNA3-HBV1.1 (HBV intact genome), HBx, HBc or preS2 Fragments over-expression plasmids. The results of PCR and Westernblot suggest that HBx can promote the expression of CUL4B1.3.2 HBx protein plays an important role in CUL4B promoting HBV replication in vitroHepG2 cells were co-transfected with CUL4B siRNA or negative control NC siRNA along with pcDNA3-HBV1.1 (HBx protein wild type group) or pcDNA3-HBV1.1 (AHBx) (HBx protein expression deficiency group) or pcDNA-HBV1.1 (AHBx) and pcDNA3-HBx-HA (HBx rescue group). The Levels of pgRNA and HBsAg of CUL4B interference group was significantly lower than control group. Suggesting that HBx protein plays an important role in CUL4B promoting HBV replication in vitro.1.3.3 In vivo experiments further verify that HBx are involved in CUL4B promoting HBV replication6-8 weeks old, male, liver-specific Cul4b knock-out mice (C57BL/6 background, Alb-Cre Cul4bFlox/Y) were injected with pcDNA3-HBV 1.1 (AHBx) (HBx protein expression deficiency group) along with pcDNA3 or pcDNA-HBV1.1 (AHBx) along with pcDNA3-HBx-HA (HBx rescue group). The Levels of HBV DNA and HBsAg of Cul4b knock-out group was significantly lower than control group. Suggesting that HBx are involved in CUL4B promoting HBV replication1.4 CUL4B can affect histone modification of HBV cccDNA minichromosome1.4.1 CUL4B and cccDNA can combined with each otherHuH-7 cells were co-transfected with prcccDNA and pCMV-Cre plasmids Results of chromatin immunoprecipitation (ChIP) show that CUL4B and cccDNA can be combined with each other1.4.2 CUL4B can promote the level of histone H3 acetylation that combined with HBV cccDNAHuH-7 cells were co-transfected with CUL4B siRNA or negative control NC siRNA and prcccDNA and pCMV-Cre plasmids. Results of ChIP-qPCR show that in the CUL4B interference group, the level of histone H3 acetylation decreased significantly, and mainly occurred on H3K.27.Meanwhile, the binding rate of cccDNA, P300 and CBP decreased significantly. It suggested that CUL4B could promote the combination of acetylated histone H3 and cccDNA.1.4.3 CUL4B can affect the level of histone H3 methylation that combined with HBV cccDNACUL4B siRNA or negative control NC siRNA were co-transfected into HepG2 or HuH-7 cells, and then pcDNA3-HBx (HBx overexpression) plasmid and control plasmid were transfected with prcccDNA and pCMV-Cre plasmid into them. Results of chromatin immunoprecipitation (ChIP) show that HBx can restore the the binding rate down-regulation of acetylated histone H3 and cccDNA after CUL4B interference. Suggesting that HBx may be involved in the regulation of the binding of histone H3 and cccDNA with CUL4B.2.Relationship between CUL4B and liver immune microenvironment2.1 Establishment of acute HBV infection model in liver-specific Cul4b knock-out mice6-8 weeks old, male, liver-specific Cul4b knock-out mice (C57BL/6 background, Alb-Cre Cul4bFlox/Y) were injected with pcDNA3-HBV1.1(HBV intact genome). The levels of HBV DNA in blood serum 24h post-injection was detected by real-time PCR.2.2 CUL4B affects the proportion of immune cells in the HBV infection modelTotal mononuclear cells (MNC) were isolated from liver. Flow cytometry was used to detect the proportion of lymphocytes in mononuclear cells. Results showed that in the control group, CUL4B had no influence on proportion and phenotypic of NK cells. However, in models of acute HBV infection, liver specific knockout of CUL4B can lead to up-regulation of IFN-γ expression on NK cells in the liver, further study show that, liver specific knockout CUL4B can also lead to up-regulation of CD69 and NKG2D expression, suggesting that CUL4B may regulate the immune microenvironment of the liver and regulate the HBV.Conclusions and significances:1. On the basis of previous work, we confirmed that HBx plays an important role in the regulation of HBV replication by CULB in vitro and vivo, while polymerase and HBc protein have no significant effect.2. This study firstly demonstrates that CUL4B can combined with HBV cccDNA, and can also regulate HBV replication by affecting the methylation/acetylation of histone。3. This study firstly find that Hepatocyte CUL4B can regulate the activation of hepatic NK cells and cytokine secretion, and then affect viral clearance after HBV infection. |
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