Both interferon and siRNA treatments promote reappearance of SMC5/6 complex but have different impact on cccDNA

Treatment with siRNA targeting HBV transcripts is currently under investigation as a novel anti-HBV treatment approach. By reducing all viral transcripts, siRNA has the potential to reduce viremia and antigenemia but has no direct effect on cccDNA stability.

Aim of this study was to investigate these antiviral effects in HBV-infected human hepatocytes in vivo and its consequences on the "structural maintenance of chromosomes" complex SMC5/6, a host restriction factor able to repress cccDNA transcription but counteracted by HBx.

Methods & experimental design:

We treated HBV-infected humanized mice for 4 to 6 weeks with siRNA targeting all viral transcripts or with pegylated interferon alpha (peg-IFNα), or mice were left untreated. Viral parameters in the serum and liver, and interferon-stimulated genes (ISG), were assessed by qPCR. cccDNA levels were also analyzed by Southern blotting. RNA in situ hybridization coupled to immunofluorescence (RNA-ISH+IF) was used to visualize both HBV transcription and SMC6.

Results:

Both treatments reduced viral RNAs and proteins, including HBx, which targets SMC5/6 for degradation, thus promoting reappearance and epigenetic binding of SMC5/6 to the cccDNA. However, only interferon treatment reduced cccDNA levels. As expected, ISG expression was elevated by IFNα but not upon siRNA treatment. SMC6 protein was degraded in the livers of untreated HBV-infected mice, but was clearly detectable in mice treated with either IFNα or siRNA. After stopping IFN, entry inhibition strategies showed that HBV rebound mostly relied on new infection events.

Conclusions:

Our study shows that both IFNα and siRNA treatment is accompanied by the reappearance of the SMC5/6 complex in human hepatocytes in vivo. This indicates that not the induction of ISGs but the suppression/reduction of HBV RNAs and related protein production is sufficient to promote SMC5/6 rebound. Although HBV RNA reduction rescued SMC5/6, targeting cccDNA stability together with entry inhibition seem necessary to cure infected cells.