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

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.

. HBx decreases the PRMT5 methylase activity and WDR77 level.
(A) HepG2 cells were transfected with Flag-tagged HBp, HBe, HBx, HBs, and HBc plasmids, respectively. The effect of HBp, HBe, HBx, HBs, and HBc on WDR77 was detected by Western 4 blot analysis in the cells 3 days later. The data are representative of two repeats.
(B) HepG2 cells were transfected with Flag-tagged HBx, HBs, HBc, HBp, and HBe plasmids, respectively. The mRNA levels of WDR77 and PRMT5 were detected by RT-qPCR in the cells 3 days later.
(C) HepG2 cells were transfected with pCH-9/3091 (WT) or HBx-deficient pCH-9/3091(△HBx) plasmids. The levels of HBx, WDR77, PRMT5 and H4R3me2s were tested by Western blot analysis in the cells 3 days later. The data are representative of two repeats.
(D) 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 in the cells 3 days later, respectively. The data are representative of two repeats. The quantification of the Western blot analysis for 3 experiments (the other test in Figure 2D) was shown (down).
(E) HEK293T cells were transfected with Flag-tagged HBx plasmids. The expression of WDR77 (red) and HBx (green) was assessed by immunofluorescence assays in the cells 3 days later.
(F) HepG2 cells were transfected with pcDNA3.1-HBx or pcDNA3.1-Vector plasmids. PRMT5 was immunoprecipitated by anti-PRMT5 antibody in the cells, and the levels of WDR77 was analyzed by CoIP analysis 3 days later. The data are representative of two repeats.
(G) HepG2-WDR77 cells were co-transfected with Flag-PRMT5 and pcDNA3.1-HBx or pcDNA3.1-Vector plasmids. An in vitro methylation assays were performed by using Flag-PRMT5 purified from the cells 3 days later. The data are representative of two repeats.
(H) HepG2 cells were transfected with pcDNA3.1-HBx or pcDNA3.1-Vector 48 h, followed by the treatment with cycloheximide (CHX) (100 μg/mL) according to the indicated time. The 5 protein levels of WDR77 and PRMT5 were detected by Western blot analysis in the cells. The data are representative of two repeats.
(F) HepG2 cells were co-transfected with Flag-tagged HBx and siCtrl or siDDB1. The Flagtagged HBx (green) and WDR77 (red) was determined by immunofluorescence staining in the cells 3 days later. Scale bars, 10 μm.
(G) HepG2-NTCP cells were infected with wild-type HBV (at a multiplicity of infection of 1000 vp/cell). CoIP assays were used to test the binding of HBx to WDR77 in the cells.