Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo

Abstract

Objective: Therapeutic strategies silencing and reducing the hepatitis B virus (HBV) reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo.

Design: HBV-infected human liver chimeric mice were treated with siRNA or peg-IFNα. Virological and host changes were analysed at the end of treatment and during the rebound phase by qualitative PCR, ELISA, immunoblotting and chromatin immunoprecipitation. RNA in situ hybridisation was combined with immunofluorescence to detect SMC6 and HBV RNAs at single cell level. The entry inhibitor myrcludex-B was used during the rebound phase to avoid new infection events.

Results: Both siRNA and peg-IFNα strongly reduced all HBV markers, including HBx levels, thus enabling the reappearance of SMC5/6 in hepatocytes that achieved HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and enhanced IFN-stimulated genes. However, the antiviral effects did not persist off treatment and SMC5/6 was again degraded. Remarkably, the blockade of viral entry that started at the end of treatment hindered renewed degradation of SMC5/6.

Conclusion: These results reveal that therapeutics abrogating all HBV transcripts including HBx promote epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA silencing.

Keywords: antiviral therapy; hepatitis B; interferon-α; liver.

Figure 1

Anti-HBV siRNA treatment potently reduces all HBV markers except cccDNA. (A) Experimental setup. HBV-infected USG mice were treated as indicated and blood was drawn every other week. (B–D) Viral titres were determined by qPCR (B), and serum HBsAg (C) and HBeAg (D) were determined by ELISA (Abbot Architect). Every line represents one mouse. (E, F) Mice were sacrificed at the indicated time points, and cccDNA levels were determined by qPCR in Epicentre-based DNA extracts without proteinase K after PSD digestion (E). cccDNA copies were normalised to the human mitochondrial gene ND2. Each dot represents a single mouse; horizontal lines depict the median. (F) Liver DNA extracts (Epicentre-based extraction without proteinase K) from D were subjected to Southern blot. DNA amounts were normalised to human mitochondrial DNA and digested with PSD before loading. The bar graph below shows the densitometry analysis of the cccDNA band. The blot depicts one representative experiment. cccDNA, covalently closed circular DNA; MyrB, myrcludex-B; NT, not treated; pf-rcDNA, protein-free relaxed circular DNA; qPCR, quantitative PCR; SB, Southern blot; siRNA, small interferring RNA; USG, uPA/SCID/beige/IL2RG^–/–. †=this mouse died prematurely at the 2-week blood drawl.

Figure 2

siRNA treatment leads to the reappearance of SMC6 in hepatocytes negative for signs of active HBV replication. (A) Immunofluorescence costaining for HBcAg (red) and SMC6 (green) in cryopreserved liver sections. Shown are representative pictures of one mouse from every treatment group as indicated on the left-hand side. (B) Representative pictures of RNA in situ hybridisation for total HBV RNA combined with immunofluorescence staining for SMC6 protein. Merged pictures of nuclei stained with 4′,6-diamidino-2-phenylindole (blue) and B2M RNA as a marker for human hepatocytes (aqua) are shown in the left column; HBV RNA (red) and SMC6 (green) are shown in the right column. Scale bar 50 µm. MyrB, myrcludex-B; siRNA, small interferring RNA.

Figure 3

Viral markers including cccDNA are reduced on peg-IFNα treatment and rebound after treatment cessation. (A) Experimental setup. HBV-infected USG mice were treated with peg-IFNα as indicated in two independent experiments. (B) Blood was drawn every other week and viral titres were determined by qPCR. The line graph shows mice from experiment 2. Lines depict the median and error bars the range. (C, D) Mice were sacrificed at the indicated time points, and liver DNA extracts (Epicentre-based extraction without proteinase K) were subjected to SB (C). DNA amounts were normalised to human mitochondrial DNA and digested with PSD before loading. The bar graph below shows the densitometry analysis of the cccDNA band. The blot shows mice from experiment 2. (D) cccDNA levels were determined by qPCR in Epicentre-based DNA extracts without proteinase K after PSD digestion in all mice (same extracts as in B). cccDNA copies were normalised to the human mitochondrial gene ND2. Each dot represents a single mouse; horizontal lines depict the median. cccDNA, covalently closed circular DNA; MyrB, myrcludex-B; NT, not treated; IFNα, interferon-α; MyrB, myrcludex-B; NT, not treated; peg-IFNα, pegylated interferon-α; pf-rcDNA, protein-free relaxed circular DNA; qPCR, quantitative PCR; SB, Southern blot; USG, uPA/SCID/beige/IL2RG^–/–.

Figure 4

Peg-IFNα treatment leads to the reappearance of the SMC5/6 complex in hepatocytes negative for signs of active replication. (A) Immunofluorescence costaining for HBcAg (red) and SMC6 (green) in cryopreserved liver sections. Shown are representative pictures of one mouse from every treatment group as indicated on the left-hand side. (B) Representative pictures of RNA in situ hybridisation for total HBV RNA combined with immunofluorescence staining for SMC6 protein. Merged pictures of nuclei stained with 4′,6-diamidino-2-phenylindole (blue) and B2M RNA as a marker for human hepatocytes (aqua) are shown in the left column; HBV RNA (red) and SMC6 (green) are shown in the right column. Scale bar 50 µm. Peg-IFNα, pegylated interferon-α; SMC5/6, structural maintenance of chromosome 5/6 complex.

Figure 5

Both siRNA and peg-IFNα treatment reduce HBx and recruit the SMC5/6 complex to the cccDNA. (A, B) HBx levels were measured in liver protein lysates after siRNA (A) and peg-IFNα (B) treatment by IP and WB using the same anti-HBx antibody (upper panel). For normalisation to human hepatocyte content, protein lysates were subjected in parallel to WB using a human-specific antibody recognising albumin (lower panel). The bar graph below shows the densitometry analysis of the HBx signal relative to human albumin. One representative picture from a total of three experiments is shown. (C, D) ChIP assays were performed in two mice from every treatment group (siRNA (C) and peg-IFNα (D)), including untreated controls and follow-up arms. Chromatin from liver tissue was precipitated with anti-NSE4 antibody and analysed by cccDNA-selective qPCR. ChIP assays were performed in triplicates for one of the mouse of every treatment group to confirm accuracy (see online supplemental figure 8). cccDNA, covalently closed circular DNA; ChIP, chromatin immunoprecipitation; IP, immunoprecipitation; MyrB, myrcludex-B; NT, not treated; peg-IFNα, pegylated interferon-α; qPCR, quantitative PCR; WB, western blot; siRNA, small interferring RNA; SMC5/6, structural maintenance of chromosome 5/6 complex.

Figure 6

Graphical summary of the main findings. Schematic representation showing cccDNA-driven production of HBV RNAs and HBx in infected human hepatocytes. Through HBx, the SMC5/6 complex is degraded, thus preventing silencing of the cccDNA. Treatment with siRNA or IFNα lowers HBV RNAs and depletes HBx in vivo, enabling reappearance of the SMC5/6 complex, its recruitment onto the cccDNA and silencing of cccDNA transcription. By combining such interventions with entry/spreading inhibition strategies, long-term suppression of the HBV minichromosome can be maintained. RNA ISH assays combined with SMC6 staining show reappearance of the host restriction factor SMC5/6 in HBV RNA-negative human hepatocytes in chimeric mice. cccDNA, covalently closed circular DNA; MyrB, myrcludex-B; IFNα, interferon-α; ISH, in situ hybridisation; siRNA, small interferring RNA; SMC5/6, structural maintenance of chromosome 5/6.