[The E3 ubiquitin ligase KLHL8 degrades HBx protein via the ubiquitin-proteasome pathway and inhibits HBV cccDNA transcription].

Objective: To screen for host factors involved in the degradation of hepatitis B virus X protein (HBx) and to investigate whether they affect hepatitis B virus (HBV) replication by modulating HBx stability. Methods: HuProt™ human proteome microarrays were used to screen for HBx-interacting proteins, combined with bioinformatics analysis focusing on E3 ligases within the ubiquitin-proteasome system. The effects of candidate proteins on HBx stability were validated by western blotting, HiBiT activity assays, cycloheximide chase assays, and proteasome inhibitor treatment. In an HBV infection model, quantitative PCR, enzyme-linked immunosorbent assay, chromatin immunoprecipitation, and micrococcal nuclease assays were employed to assess the effects of the E3 ligase on the transcriptional activity of covalently closed circular DNA (cccDNA), viral replication, and epigenetic status. Intergroup comparisons were performed using one-way ANOVA, Student's t-test, or the non-parametric Mann-Whitney U test as appropriate. Results: A total of 290 HBx-interacting candidate proteins were identified. Among them, the ubiquitin-proteasome system-related proteins Kelch-like protein 8 (KLHL8) and the canonical HECT-type E3 ligase WWP2 significantly reduced HBx protein levels and, more notably, suppressed cccDNA-mediated viral transcription. Cycloheximide chase assays demonstrated that KLHL8 overexpression markedly shortened the half-life of HBx, and this effect was reversed by a 26S proteasome inhibitor, whereas WWP2 had a comparatively limited effect on the degradation rate of HBx. Functional experiments showed that KLHL8 overexpression inhibited cccDNA transcriptional activity and viral replication, reduced HBx binding to cccDNA, and decreased chromatin accessibility. Conclusion: The E3 ubiquitin ligase KLHL8 degrades HBx through the ubiquitin-proteasome pathway, thereby inhibiting HBV cccDNA transcription and replication. These findings provide a theoretical basis for novel anti-HBV strategies targeting the host ubiquitination machinery.