Background & aims: HBV persistence is maintained by both an episomal covalently closed circular (ccc)DNA reservoir and genomic integration of HBV DNA fragments. While cccDNA transcription is regulated by Cullin4A-DDB1-HBx-mediated degradation of the SMC5/6 complex, HBsAg expression from integrants is largely SMC5/6 independent. Inhibiting neddylation of Cullin-RING ubiquitin ligases impairs degradation of substrates. Herein, we show that targeting neddylation pathway components by small-interfering (si)RNAs or the drug MLN4924 (pevonedistat) suppresses expression of HBV proteins from both cccDNA and integrants.
Methods: An siRNA screen targeting secretory pathway regulators and neddylation genes was performed. Activity of MLN4924 was assessed in infection and integration models. Trans-complementation assays were used to study HBx function in cccDNA-driven expression.
Results: siRNA screening uncovered neddylation pathway components (Nedd8, Ube2m) that promote HBsAg production post-transcriptionally. Likewise, MLN4924 inhibited production of HBsAg encoded by integrants and reduced intracellular HBsAg levels, independent of HBx. MLN4924 also profoundly inhibited cccDNA transcription in three infection models. Using the HBV inducible cell line HepAD38 as a model, we verified the dual action of MLN4924 on both cccDNA and integrants with sustained suppression of HBV markers during 42 days of treatment.
Conclusions: Neddylation is required both for transcription of a cccDNA reservoir and for the genomic integration of viral DNA. Therefore, blocking neddylation might offer an attractive approach towards functional cure of chronic hepatitis B.
Lay summary: Current treatments for chronic hepatitis B are rarely able to induce a functional cure. This is partly because of the presence of a pool of circular viral DNA in the host nucleus, as well as viral DNA fragments that are integrated into the host genome. Herein, we show that a host biological pathway called neddylation could play a key role in infection and viral DNA integration. Inhibiting this pathway could hold therapeutic promise for patients with chronic hepatitis B.
Keywords: DDB1, DNA damage-binding protein 1; HBsAg; HBsAg, hepatitis B virus surface antigen; HBx; HBx, hepatitis B virus X protein; MLN4924; NAE1, NEDD8-activating enzyme E1 subunit 1; NEDD8, neural precursor cell expressed, developmentally downregulated 8; PHHs, primary human hepatocytes; SMC6; SVP, subviral particles; Smc5/6, structural maintenance of chromosomes 5/6; WT, wild-type; cccDNA; cccDNA, covalently closed circular DNA; integrants; neddylation; pgRNA, pregenomic RNA; siRNA, small-interfering RNA; transcription.
© 2022 The Author(s).