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. 2021 Jul 25;11(17):8362-8378.
doi: 10.7150/thno.57531. eCollection 2021.

HBx represses WDR77 to enhance HBV replication by DDB1-mediated WDR77 degradation in the liver

Hongfeng Yuan  1   2 , Lina Zhao  2 , Ying Yuan  2 , Haolin Yun  2 , Wei Zheng  2 , Yu Geng  2 , Guang Yang  1 , Yufei Wang  2 , Man Zhao  2 , Xiaodong Zhang  1
Affiliations

HBx represses WDR77 to enhance HBV replication by DDB1-mediated WDR77 degradation in the liver

Hongfeng Yuan et al. Theranostics. .

Abstract

Rationale: Hepatitis B x protein (HBx) is required to initiate and maintain the replication of hepatitis B virus (HBV). Protein arginine methyltransferases 5 (PRMT5) negatively regulates HBV transcription. WD repeat domain 77 protein (WDR77) greatly enhances the methyltransferase activity of PRMT5. However, the role of WDR77 in the modulation of cccDNA transcription and HBV replication is poorly understood. In this study, we investigated the mechanism by which HBx modulated HBV replication involving WDR77 in the liver. Methods: A human liver-chimeric mouse model was established. Immunohistochemistry (IHC) staining, Western blot analysis, Southern blot analysis, Northern blot analysis, immunofluorescence assays, ELISA, RT-qPCR, CoIP assays, and ChIP assays were performed in human liver-chimeric mouse model, primary human hepatocytes (PHHs), HepG2-NTCP, dHepaRG and HepG2 cell lines. Results: HBV infection and HBx expression remarkably reduced the protein levels of WDR77 in human liver-chimeric mice and HepG2-NTCP cells. WDR77 restricted cccDNA transcription and HBV replication in PHHs and HepG2-NTCP cells. Mechanically, WDR77 enhanced PRMT5-triggered symmetric dimethylation of arginine 3 on H4 (H4R3me2s) on the cccDNA minichromosome to control cccDNA transcription. HBx drove the cellular DDB1-containing E3 ubiquitin ligase to degrade WDR77 through recruiting WDR77, leading to the disability of methyltransferase activity of PRMT5. Thus, HBx promoted HBV replication by driving a positive feedback loop of HBx-DDB1/WDR77/PRMT5/H4R3me2s/cccDNA/HBV/HBx in the liver. Conclusions: HBx attenuates the WDR77-mediated HBV repression by driving DDB1-induced WDR77 degradation in the liver. Our finding provides new insights into the mechanism by which HBx enhances HBV replication in the liver.

Keywords: DDB1; H4R3me2s; HBx; PRMT5; WDR77.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
HBV infection leads to the decrease of PRMT5 methylase activity and WDR77 level. (A) A model of establishment of human liver-chimeric mice. (B) Immunohistochemistry assays for HBcAg and WDR77 were performed in the liver tissues from human liver-chimeric mice. N = 3 in each group. Scale bars: 50 μm. (C) The levels of H4R3me2s, Rme2sy, PRMT5, and WDR77 were examined by Western blot analysis in the liver tissues from human liver-chimeric mice. The quantification of the Western blot analysis for 3 experiments was shown (down). The other two Western tests were shown in Figure S1B. (D) HepG2-NTCP cells were uninfected or infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell). The levels of H4R3me2s, Rme2sy, PRMT5, and WDR77 were tested by Western blot analysis 7 days later. The quantification of the Western blot analysis for 3 experiments was shown (down). The other two Western tests were shown in Figure S1C. (E) HepG2 cells were transfected with pCH9 (vector control, 2 μg) or pCH9/3091 (HBV-expressing plasmid, 2 μg) plasmids. The levels of H4R3me2s, Rme2sy, PRMT5, and WDR77 were evaluated by Western blot analysis 3 days later. The quantification of Western blot analysis for 3 experiments was shown (down). The other two Western tests were shown in Figure S1D. (F) HepG2-NTCP cells were uninfected or infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell). The expression of HBcAg and WDR77 was assessed by immunofluorescence assays 7 days later. Scale bars: 10 μm. (G) A model of HBV reducing the methyltransferase activity of PRMT5.
Figure 2
Figure 2
HBx decreases the PRMT5 methylase activity and WDR77 level. (A) HepG2 cells were transfected with Flag-tagged HBp, HBe, HBx, HBs, and HBc plasmids (2 μg), respectively. The effect of HBp, HBe, HBx, HBs, and HBc on WDR77 was detected by Western blot analysis 3 days later. (B) HepG2 cells were transfected with pCH-9/3091 (WT, 2 μg) or HBx-deficient pCH-9/3091(ΔHBx, 2 μg) plasmids. The levels of HBx, WDR77, PRMT5, and H4R3me2s were tested by Western blot analysis 3 days later. (C) HepG2 cells were transfected with pcDNA3.1-HBx (2 μg) or pcDNA3.1-Vector plasmids (2 μg). The levels of HBx, WDR77, PRMT5, and H4R3me2s were measured by Western blot analysis 3 days later. (D) HepG2 cells were transfected with Flag-tagged HBx plasmids (2 μg). The expression of WDR77 (red) and HBx (green) was assessed by immunofluorescence assays 3 days later. (E) The levels of HBx, WDR77, PRMT5, DDB1 and H4R3me2s were examined by Western blot analysis in the clinical needle biopsy liver tissues. (F) HepG2 cells were transfected with pcDNA3.1-HBx (2 μg) or pcDNA3.1-Vector (2 μg) plasmids. PRMT5 was immunoprecipitated by anti-PRMT5 antibody from the cells, and the levels of WDR77 were analyzed by CoIP analysis 3 days later. (G) HepG2-WDR77 cells were co-transfected with Flag-PRMT5 and pcDNA3.1-HBx (2 μg) or pcDNA3.1-Vector (2 μg) plasmids. An in vitro methylation assays were performed by using Flag-PRMT5 purified from the cells 3 days later. (H) HepG2 cells were transfected with pcDNA3.1-HBx or pcDNA3.1-Vector 48 h followed by cycloheximide (CHX) (100 μg/mL) treatment for the indicated time. The protein levels of WDR77 and PRMT5 were detected by Western blot analysis. The quantitative analysis of WDR77 and PRMT5 expression was shown (right).
Figure 3
Figure 3
WDR77 represses the HBV replication. (A) Schematic representation of cell experimental process. (B) PHH cells were infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell) and were continuously transfected with indicated plasmids or siWDR77 (siWDR77#3) at -2, 1, and 4 dpi (days post-infection). The levels of WDR77 were assessed by Western blot analysis 7 days post-infection. (C) The levels of HBV DNA were measured by qPCR in the supernatant of HBV de novo infection PHH cells transfected with siWDR77 or pcDNA3.1-WDR77. (D) The levels of HBsAg were examined by ELISA in the supernatant of HBV de novo infection PHH cells transfected with siWDR77 or pcDNA3.1-WDR77. (E) The levels of HBeAg were determined by ELISA in the supernatant of HBV de novo infection PHH cells transfected with siWDR77 or pcDNA3.1-WDR77. (F) The expression of HBc (green) and WDR77 (red) was determined by immunofluorescence staining in HBV de novo infection PHH cells transfected with pcDNA3.1-WDR77 (2 μg). Scale bars, 10 μm. (G) PHH cells were infected with HBx-deficient HBV (ΔX, at a multiplicity of infection of 1000 vp/cell) and were continuously transfected with indicated plasmids or siRNA of WDR77 at -2, 1, and 4 dpi (days post-infection). The levels of HBV DNA, HBsAg and HBeAg were measured by qPCR and ELISA in the supernatant of the cells 7 days post-infection. (H) The levels of HBV DNA were determined by qPCR in the supernatant of HBx-deficient HBV de novo infection PHH cells (ΔX) transfected with siCtrl (100 nM), siWDR77 (100 nM), pcDNA3.1-Vector (2 μg), or pcDNA3.1-WDR77 (WDR77-OE, 2 μg). (I) The levels of HBsAg and HBeAg were examined by ELISA in the supernatant of HBx-deficient HBV de novo infection PHH cells (ΔX) transfected with siCtrl (100 nM), siWDR77 (100 nM), pcDNA3.1-Vector (2 μg), or pcDNA3.1-WDR77 (WDR77-OE, 2 μg). Data are represented as means ± SD (n = 3). Student's t test, *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4
Figure 4
WDR77 attenuates the transcription activity of cccDNA. (A) PHH cells were infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell) and were continuously transfected with plasmids or siRNA of WDR77 at -2, 1, and 4 dpi (days post-infection). The levels of HBV pgRNA were quantified by RT-qPCR assays 7 days post-infection. (B) PHH cells were infected with wild-type or HBx-deficient HBV (at a multiplicity of infection of 1000 vp/cell) and were continuously transfected with pcDNA3.1-HBx (10 μg), siWDR77 (100 nM) or pcDNA3.1-WDR77 (10 μg) at -2, 1, and 4 dpi (days post-infection). The levels of HBV RNA were tested by Northern blot analysis 7 days post-infection. (C) PHH cells were infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell) and were continuously transfected with siWDR77 or pcDNA3.1-WDR77 at -2, 1, and 4 dpi (days post-infection). The levels of cccDNA were evaluated by RT-qPCR analysis 7 days post-infection. (D) PHH cells were infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell) and were continuously transfected with siWDR77 (100 nM) or pcDNA3.1-WDR77 (10 μg) at -2, 1, and 4 dpi (days post-infection). The levels of cccDNA were tested by Southern blot analysis 7 days post-infection. (E) HBV transcription activity was assessed by calculating the ratio of pgRNA/cccDNA. (F) A model of WDR77 reducing the transcription activity of cccDNA. Data are represented as means ± SD (n = 3). Student's t test, ns, no significant; *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 5
Figure 5
WDR77 is required for the PRMT5-mediated inhibition of cccDNA transcription. (A) HepG2-NTCP cells were infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell) and were continuously transfected with siWDR77 (100 nM) or pcDNA3.1-WDR77 (2 μg) at -2, 1, and 4 dpi (days post-infection). The levels of Rme2sy, WDR77, PRMT5 and H4R3me2s were detected by Western blot analysis 7 days post-infection. (B) HepG2-NTCP cells were infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell) and were continuously transfected with siPRMT5 (100 nM) or pcDNA3.1-WDR77 (2 μg) at -2, 1, and 4 dpi (days post-infection). The levels of Rme2sy, WDR77, PRMT5 and H4R3me2s were evaluated by Western blot analysis 7 days post-infection. (C) HBV transcription activity was assessed by calculating the ratio of pgRNA/cccDNA in HBV de novo infection HepG2-NTCP cells at the indicated days after HBV infection. (D) The assembly of H4R3me2s onto cccDNA was examined by ChIP-qPCR in HBV de novo infection HepG2-NTCP cells in the indicated days after HBV infection. (E) HepG2-NTCP cells were infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell). The assembly of PRMT5 or WDR77 onto cccDNA was determined by ChIP-qPCR 7 days later. (F) HepG2-NTCP cells were infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell). Re-ChIP assay was performed with the indicated antibodies 7 days later. (G) The assembly of H4R3me2s onto cccDNA was determined by ChIP-qPCR in HBV de novo infection HepG2-NTCP cells transfected with siWDR77 (100 nM), pcDNA3.1-WDR77 (10 μg) or co-transfected with pcDNA3.1-WDR77 (10 μg) and siPRMT5 (100 nM). (H) HBV transcription activity was evaluated by calculating the ratio of pgRNA/cccDNA in HBV de novo infection HepG2-NTCP cells transfected with siWDR77 (100 nM), pcDNA3.1-WDR77 (10 μg) or co-transfected with pcDNA3.1-WDR77 (100 nM) and siPRMT5 (10 μg). (I) The assembly of H4R3me2s onto cccDNA was determined by ChIP-qPCR in HBV de novo infection dHepaRG cells transfected with siWDR77 (100 nM), pcDNA3.1-WDR77 (10 μg) or co-transfected with pcDNA3.1-WDR77 (10 μg) and siPRMT5 (100 nM). (J) HBV transcription activity was evaluated by calculating the ratio of pgRNA/cccDNA in HBV de novo infection dHepaRG cells transfected with siWDR77 (100 nM), pcDNA3.1-WDR77 (10 μg) or co-transfected with pcDNA3.1-WDR77 (10 μg) and siPRMT5 (100 nM). (K) A model of WDR77 suppressing cccDNA transcription based on PRMT5 methyltransferase activity. Data are represented as means ± SD (n = 3). Student's t test, *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 6
Figure 6
HBx degrades WDR77 by DDB1-CUL4-ROC1 E3 ligase. (A) HepG2 cells were transfected with Flag-tagged HBx (2 μg) for 48 h and then treated with MG132 (2 mM) for 24 h. The protein levels of WDR77 were detected by Western blot analysis in the cells. (B) HepG2 cells were transfected with the indicated plasmids (pcDNA3.1-HBx and HA-tagged Ubiquitin, 10 μg) and treated with MG132 (2 mM) 4 h before harvest. The ubiquitylation (Ub) of immunoprecipitated WDR77 was examined by Western blot analysis in the cells 3 days later. (C) HepG2 cells were transfected with HA-tagged HBx (2 μg) and/or siDDB1 (100 nM). The protein levels of WDR77, HBx and DDB1 were measured by Western blot analysis in the cells 3 days later. (D) His-tagged HBx was purified from E.coli with Ni-NTA resin. Flag-tagged WDR77 was purified by anti-DDDDK-tag mAb-magnetic agarose from HEK293T cells transfected with Flag-tagged WDR77 (10 μg). The beads with Flag-WDR77 were incubated with His-HBx and examined by immunoblotting with anti-His antibody. (E) HepG2 cells were co-transfected with Flag-tagged HBx (2 μg), HA-tagged WDR77 (2 μg), and siDDB1 (100 nM). The co-localization of exogenous Flag-tagged HBx (green) and HA-tagged WDR77 (red) were determined by immunofluorescence staining with confocal microscopy 3 days later. Scale bars, 10 μm. (F) HepG2 cells were transfected with HA-tagged HBx (10 μg) and/or siDDB1 (100 nM). The binding of HBx to immunoprecipitated WDR77 was examined by Western blot analysis 3 days later. (G) HepG2 cells were co-transfected with pcDNA3.1-HBx (10 μg), HA-tagged Ubiquitin (10 μg) and siDDB1 (100 nM), followed by treatment with MG132 (2 mM) 4 h before harvest. Ubiquitylation of immunoprecipitated WDR77 was tested by Western blot analysis 3 days later. (H) HepG2 cells were co-transfected with pcDNA3.1-HBx/pcDNA3.1-HBx mut (10 μg), HA-tagged Ubiquitin (10 μg) and Flag-tagged WDR77 (10 μg), followed by treatment with MG132 (2 mM) 4 h before harvest. Ubiquitylation of immunoprecipitated WDR77 was evaluated by Western blot analysis 3 days later. (I) A model of HBx targeting WDR77 for ubiquitylation by DDB1-ROC1 E3 ligase. a, HBx binds to DDB1 and recruits WDR77 for ubiquitylation by DDB1-ROC1 E3 ligase. b, HBx mutant failed to bind to DDB1 and DDB1-ROC1 E3 ligase cannot work.
Figure 7
Figure 7
A model of HBx degrading WDR77 to enhance HBV replication by DDB1-mediated WDR77 degradation in the liver. In this model, HBx binds to DDB1 and recruits WDR77 for degradation by the DDB1-CUL4-ROC1 (CRL4) E3 ligase. WDR77 is able to enhance the methylase activity of PRMT5 which inhibits cccDNA transcription by increasing the cccDNA-bound H4R3me2s. The degradation of WDR77 mediated by HBx-DDB1 leads to the decrease of the cccDNA-bound H4R3me2s catalyzed by PRMT5, resulting in the activation of cccDNA transcription.
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