Dual role of neddylation in transcription of hepatitis B virus RNAs from cccDNA and production of viral surface antigen
Bingqian Qu,
Firat Nebioglu,
Mila M. Leuthold,
Yi Ni,
Pascal Mutz,
Jürgen Beneke,
Holger Erfle,
Florian W.R. Vondran,
Ralf Bartenschlager,
Stephan Urban
Affiliations
Bingqian Qu
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; Division of Veterinary Medicine, Paul Ehrlich Institute, Langen, Germany
Firat Nebioglu
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
Mila M. Leuthold
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
Yi Ni
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; German Center for Infection Research (DZIF), Heidelberg Partner site, Heidelberg, Germany
Pascal Mutz
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
Jürgen Beneke
Advanced Biological Screening Facility Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), Heidelberg University, Heidelberg, Germany
Holger Erfle
Advanced Biological Screening Facility Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), Heidelberg University, Heidelberg, Germany
Florian W.R. Vondran
German Center for Infection Research (DZIF), Hannover-Braunschweig Partner site, Hannover, Germany; Regenerative Medicine and Experimental Surgery (ReMediES), Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany
Ralf Bartenschlager
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany; German Center for Infection Research (DZIF), Heidelberg Partner site, Heidelberg, Germany; Corresponding authors. Address: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; Tel.: +49-(0)6221-56-4902, fax: +49-(0)6221-56-1946.
Stephan Urban
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; German Center for Infection Research (DZIF), Heidelberg Partner site, Heidelberg, Germany; Corresponding authors. Address: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; Tel.: +49-(0)6221-56-4902, fax: +49-(0)6221-56-1946.
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.
