Objective: Hepatitis B virus (HBV) infection is a major public health problem worldwide. However, the regulatory mechanisms underlying HBV replication remain unclear. Cullin 4B-RING ubiquitin E3 ligase (CRL4B) is involved in regulating diverse physiological and pathophysiological processes. In our study, we aimed to explain the role of CUL4B in HBV infection. Methods: Cul4b transgenic mice or conditional knockout mice, as well as liver cell lines with CUL4B overexpression or knockdown, were used to assess the role of CUL4B in HBV replication. Immunoprecipitation assays and immunofluorescence staining were performed to study the interaction between CUL4B and HBx. Cycloheximide chase assays and in vivo ubiquitination assays were performed to evaluate the half-life and the ubiquitination status of HBx. Results: The hydrodynamics-based hepatitis B model in Cul4b transgenic or conditional knockout mice indicated that CUL4B promoted HBV replication (P < 0.05). Moreover, the overexpression or knockdown system in human liver cell lines validated that CUL4B increased HBV replication in an HBx-dependent manner. Importantly, immunoprecipitation assays and immunofluorescence staining showed an interaction between CUL4B and HBx. Furthermore, CUL4B upregulated HBx protein levels by inhibiting HBx ubiquitination and proteasomal degradation (P < 0.05). Finally, a positive correlation between CUL4B expression and HBV pgRNA level was observed in liver tissues from HBV-positive patients and HBV transgenic mice. Conclusions: CUL4B enhances HBV replication by interacting with HBx and disrupting its ubiquitin-dependent proteasomal degradation. CUL4B may therefore be a potential target for anti-HBV therapy.

Key Laboratory for Experimental Teratology of the Ministry of Education,Key Laboratory of Infection and Immunity of Shandong Province and Department of Immunology,School of Basic Medical Sciences,Shandong University,Jinan 250012,China;Department of Laboratory Diagnosis,Cangzhou Combination of Traditional Chinese and Western Medicine Hospital of Hebei Province,Cangzhou 061000,China;Division of Gastroenterology and Hepatology,Renji Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai Institute of Digestive Disease,Shanghai 200120,China;Ministry of Education Key Laboratory of Experimental Teratology and Institute of Molecular Medicine and Genetics,School of Basic Medical Sciences,Shandong University,Jinan 250012,China;Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong,Jinan 250012,China;Department of Gastroenterology,Qilu Hospital,Shandong University,Jinan 250012,China

[1]Haixia Shan;Bo Wang;Xiaodong Zhang;Hui Song;Xi Li;Yongxin Zou;Baichun Jiang;Huili Hu;Hao Dou;Changshun Shao;Lifen Gao;Chunhong Ma;Xiaoyun Yang;Xiaohong Liang;Yaoqin Gong-.CUL4B facilitates HBV replication by promoting HBx stabilization)[J].癌症生物学与医学（英文版）,2022(01):120-131

CUL4B,CRL4B,Cul4b

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