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Therapeutic Shutdown of HBV Transcripts Promotes Reappearance

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Therapeutic Shutdown of HBV Transcripts Promotes Reappearance Hepatology Original research Therapeutic shutdown of HBV transcripts promotes Gut: first published as 10.1136/gutjnl-2020-322571 on 28 January 2021. Downloaded from reappearance of the SMC5/6 complex and silencing of the viral genome in vivo Lena Allweiss,1 Katja Giersch,1 Andrea Pirosu,1 Tassilo Volz ,1,2 Robert C Muench,3 Rudolf K Beran,3 Stephan Urban,2,4 Hassan Javanbakht,3 Simon P Fletcher,3 Marc Lütgehetmann,2,5 Maura Dandri 1,2 ► Additional material is ABSTRACT Significance of this study published online only. To view, Objective Therapeutic strategies silencing and reducing please visit the journal online the hepatitis B virus (HBV) reservoir, the covalently (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2020- 322571). closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the ► There is an urgent need for therapeutic 1Department of Internal impact of small interferring RNA (siRNA) targeting all approaches repressing covalently closed Medicine, University Medical circular DNA (cccDNA) activity because Center Hamburg- Eppendorf, HBV transcripts or pegylated interferon-α (peg- IFNα) Hamburg, Germany on the viral regulatory HBx protein and the structural silencing of the viral reservoir may promote 2German Center for Infection maintenance of chromosome 5/6 complex (SMC5/6), functional HBV cure. The host restriction Research (DZIF), Hamburg- a host factor suppressing cccDNA transcription. In factor structural maintenance of chromosome Lübeck- Borstel- Riems and 5/6 (SMC5/6) hinders transcription from the Heidelberg sites, Germany particular, we assessed whether interventions lowering 3Gilead Sciences Inc, Foster City, HBV transcripts can achieve and maintain silencing of HBV minichromosome, but the viral protein California, USA cccDNA transcription in vivo. HBx triggers its degradation. The impact 4 Department of Infectious Design HBV- infected human liver chimeric mice were of treatments lowering HBV transcripts on Diseases, Molecular Virology, treated with siRNA or peg- IFNα. Virological and host HBx, SMC5/6 and cccDNA activity in vivo is University Hospital Heidelberg, unknown. Heidelberg, Germany changes were analysed at the end of treatment and 5 Department of Medical during the rebound phase by qualitative PCR, ELISA, What are the new findings? Microbiology, Virology and immunoblotting and chromatin immunoprecipitation. ► RNA interference targeting all HBV transcripts Hygiene, University Medical RNA in situ hybridisation was combined with Center Hamburg- Eppendorf, and pegylated interferon-α abrogate Hamburg, Germany immunofluorescence to detect SMC6 and HBV RNAs at HBx production in a substantial amount http://gut.bmj.com/ single cell level. The entry inhibitor myrcludex-B was used of hepatocytes, leading to the prompt Correspondence to during the rebound phase to avoid new infection events. reappearance of SMC5/6 and cccDNA silencing Dr Maura Dandri, Department Results Both siRNA and peg-IFN α strongly reduced in vivo. Viral entry inhibition appears required of Internal Medicine, University all HBV markers, including HBx levels, thus enabling the to prevent cccDNA reactivation in cells that Medical Center Hamburg- reappearance of SMC5/6 in hepatocytes that achieved Eppendorf, Hamburg 20251, achieved silencing. Germany; m. dandri@ uke. de HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and How might it impact on clinical practice in the on October 2, 2021 by guest. Protected copyright. LA and KG contributed equally. enhanced IFN-stimulated genes. However, the antiviral foreseeable future? effects did not persist off treatment and SMC5/6 was ► Sustained silencing of cccDNA transcription Received 23 July 2020 Revised 12 January 2021 again degraded. Remarkably, the blockade of viral entry appears achievable by combining interventions Accepted 14 January 2021 that started at the end of treatment hindered renewed both abolishing HBx and shielding the degradation of SMC5/6. hepatocytes from new infections. These findings Conclusion These results reveal that therapeutics can assist in the design of future clinical trials abrogating all HBV transcripts including HBx promote aiming to achieve a functional cure. epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA carcinoma.1 Both the lack of robust antiviral immu- © Author(s) (or their silencing. nity and the persistence of an episomal HBV DNA employer(s)) 2021. Re- use genome, the so- called covalently closed circular permitted under CC BY- NC. No DNA (cccDNA), are held responsible for the failure commercial re- use. See rights 2 and permissions. Published of viral clearance in CHB. by BMJ. INTRODUCTION The viral genome consists of a partially double- HBV is a small enveloped DNA virus that causes stranded relaxed circular DNA that is transferred To cite: Allweiss L, Giersch K, acute and chronic infections of the liver. Chronic to the hepatocyte nucleus and converted into a Pirosu A, et al. Gut Epub ahead of print: [please hepatitis B (CHB) affects approximately 250 million stable cccDNA minichromosome, associated with 3 include Day Month Year]. individuals worldwide, causing more than 800 000 viral and host proteins similar to host chromatin. doi:10.1136/ deaths every year due to complications of liver The cellular transcriptional machinery generates gutjnl-2020-322571 disease such as cirrhosis and hepatocellular all viral transcripts needed for viral replication. An Allweiss L, et al. Gut 2021;0:1–10. doi:10.1136/gutjnl-2020-322571 1 Hepatology overlength pregenomic RNA (pgRNA) is reverse transcribed after virus inoculation). The siRNA targets all HBV transcripts by the viral polymerase within the nucleocapsids before being and was described earlier20 (referred to as W7.5 in the original enveloped and secreted as progeny virus. The infected hepato- publication). Mice received one intravenous injection of 1 mg/kg Gut: first published as 10.1136/gutjnl-2020-322571 on 28 January 2021. Downloaded from cytes also secrete the E antigen (HBeAg) and high amounts of per week. The siRNA was obtained from Dharmacon (Lafayette, subviral particles containing the envelope proteins (HBsAg). Colorado, USA) and formulated in lipid nanoparticles (LNPs) at High levels of circulating antigens are thought to contribute Precision Nanosystems (Vancouver, British Columbia, Canada). to immune tolerance and HBV persistence.4 In addition to the Peg- IFNα (Pegasys; Hoffmann- La Roche, Basel, Switzerland) capsid protein (HBcAg) and the viral polymerase, HBV produces was administered two times per week intraperitoneally (25 ng/g the regulatory X protein (HBx), which can interfere with several body weight) for 6 weeks.27 Mice were either sacrificed 24 hours cellular pathways and transcription factors, and is recruited after the last injection or left untreated for follow-up. At the end onto the cccDNA.5 6 Studies with HBV X- minus mutant viruses of siRNA or peg- IFNα treatment, some mice received subcu- demonstrated that HBx is essential for cccDNA-driven viral taneous injections of MyrB (MyrB/bulevirtide; MYR Pharma- transcription.7 8 Moreover, HBx was shown to mediate the ceuticals, Bad Homburg, Germany) at 2 µg/g body weight daily degradation of the structural maintenance of chromosome 5/6 for additional 3–8 weeks.33 Additional uninfected mice, either complex (SMC5/6),9 10 a multifunctional DNA- binding complex untreated or treated with peg- IFNα for 4 weeks,27 served as required for chromosome dynamics and stability.11 By binding controls for SMC6 immunofluorescence. to the damaged DNA-binding protein 1 (DDB1), an adaptor protein for the E3 ubiquitin ligase complex,12 HBx promotes the Immunoprecipitation/western blot interaction of SMC5/6 with the E3 ubiquitin ligase proteasome Proteins were extracted from liver tissues with T- Per Tissue system, triggering SMC5/6 degradation.13 Various viruses can Protein Extraction Reagent (Pierce, Rockford, Illinois, USA) hijack the DDB1–E3 ubiquitin ligase complex to promote the supplemented with Complete Protease Inhibitor Cocktail degradation of host proteins that would otherwise restrict viral (Roche) and Pefabloc SC-P rotease- Inhibitor (Carl Roth, Karl- replication.14 The SMC5/6 antagonism by HBx seems to be an sruhe, Germany). For immunoprecipitation, dynabeads protein evolutionarily conserved mechanism among mammalian hepad- G (Thermo Fisher) were incubated with 10 µg of monoclonal naviruses.15 SMC5/6 colocalises with the nuclear domain 10,16 rabbit anti- HBx antibody34 for 10 min at room tempera- a dynamic nuclear organelle also implicated in the restriction of ture. Protein concentrations were determined by BCA Protein DNA viruses.17 It is thus not surprising that the SMC5/6 complex Assay Kit (Pierce), and 1 mg of protein lysate was added to the can act as a host restriction factor in HBV infection. Despite dynabeads antibody complex and incubated overnight at 4°C. the increasing awareness of the role of SMC5/6 in counteracting Washing, denaturing and elution were performed according cccDNA activity, data on how antiviral treatments affect the to the manufacturer’s instructions. Proteins were resolved on interaction between HBx and SMC5/6 in vivo are absent. 12% Mini-PROTEAN TGX Precast Gels (Bio-Rad, Feldkirchen, Potent nucleos(t)ide analogues efficiently suppress viral Germany) and blotted on nitrocellulose membranes (0.2
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