CUL4B Facilitates HBV Replication by Promoting HBx Stabilization

Background: Cullin 4B-RING ubiquitin E3 ligase (CRL4B) is involved in regulating diverse physiological and pathophysiological processes. However, the role of CUL4B in hepatitis B virus (HBV) infection remains completely unknown. Methods: Cul4b transgenic mice or conditional knockout mice, as well as liver cell lines with CUL4B overexpression or knockdown, were used for estimating the role of CUL4B in HBV replication. HBx-null and polymerase-null HBV plasmids were introduced to assess the involvement of viral proteins in CUL4B regulation on HBV replication. Immunoprecipitation assay and immunofluorescence staining were performed to study the interaction between CUL4B and HBx. Cycloheximide (CHX) chase assay and in vivo ubiquitination assay was performed to evaluate the half-life of HBx protein and the ubiquitination status of HBx. Findings: The hydrodynamics-based hepatits B model in Cul4b transgenic or conditional knockout mice indicated the promotion of CUL4B on HBV replication.  Consistently, the overexpression or knockdown system in human liver cell lines validated that CUL4B improved HBV replication in a HBx-dependent manner. Importantly, the immunoprecipitation assay and the immunofluorescence staining showed the interaction between CUL4B and HBx.  Furthermore, CUL4B upregulated HBx protein level and increased the half-life of HBx by inhibiting its ubiquitination and proteasomal degradation. Finally, the positive correlations between CUL4B expression and HBV pgRNA level were observed in liver tissues from HBV-positive patients or HBV transgenic mice. Interpretation: CUL4B enhances HBV replication by interacting with HBx and disrupting its ubiquitin-dependent proteasomal degradation. Interference of CUL4B represents a potential target for anti-HBV therapy. Funding: This work was supported by grants from the National Science Foundation of China (No. 81970508, 81672425, 818300178, 8197148, 81670520), the National Key Research and Development Program (No. 2018YFE0126500, 2016YFE0127000), Key Research & Development Plan of Shandong Province (No. 2018YFJH0503, 2016ZDJS07A17, 2017GSF18185). Thanks for the supporting from Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong. Declaration of Interests: No conflict of interest exists in the submission of this manuscript, and manuscript is approved by all authors for publication. Ethics Approval Statement: Informed consent was obtained from all patients before the study was performed with the approval of Shandong University Medical Ethics Committee in accordance with the Declaration of Helsinki. All animal procedures were in compliance with national regulations and approved by Animal Use Committee, Shandong University School of Medicine.