Degradation by APOBECs
Many of the IFN subtypes with anti-HBV properties induce APOBEC family genes, which are cytidine deaminases that perform RNA editing. They have also been well-documented to act directly on cccDNA to result in degradation. IFN-α, which is used in clinical therapy of CHB, has been shown to reduce the expression of cccDNA, HBV RNA, HBs and HBe in primary human hepatocytes (PHH) and HepRG liver cells. The IFN-α mediated loss of cccDNA was found to last up to 15 d. Induced expression of APOBECs can be seen after IFN treatment in liver biopsies of hepatitis B patients, and in HBV-infected chimpanzees. The importance of APOBECs in mediating cccDNA clearance is supported by the fact that the expression of APOBECs correlates with clinical response to IFN.
Besides IFN-α and IFN-γ, other pathways induced by pro-inflammatory cytokines including TNF-α and lymphotoxin-β (LT-β) have also been shown to induce APOBECs and affect the stability of cccDNA. In agreement with its anti-cccDNA functions, APOBECs induced through LT-β signalling with LTβR agonist antibody was shown to clear 90% of cccDNA in HBV-infected cultured liver cells with good in vivo tolerability. As the clinical dose of IFN-α used to achieve cccDNA clearance is relatively high, this suggests that inducing expression of APOBECs through LT-β signalling is an alternative means for cccDNA clearance with potentially fewer adverse effects. While its clinical efficacy and safety remain to be studied, upregulation of natural ligands for LTβR in HBV-infected liver tissues have been reported. IFN-γ and TNF-α also exhibit anti-cccDNA effects to degrade cccDNA levels without causing cell death in HBV-infected chimpanzees, PHH and cultured liver cells.
Following IFN receptor or LTβR activation, the upregulated IFN-α induced APOBEC3A (A3A) and LTβR activation-induced APOBEC3B (A3B) are brought to cccDNA by HBc (Figure 2). Similarly, IFN-γ induced the expression of both A3A and A3B while TNF-α induced the expression of A3B only, whereas knockdown of A3A and A3B abrogated the antiviral effects of IFN-γ and TNF-α. HBc amino acids 77-149 are crucial for this interaction, which brings the APOBECs into close proximity with cccDNA for deamination. cccDNA deamination generates apurinic/apyrimidinic (AP) sites that are recognized by endonucleases, which ultimately degrade cccDNA. The dependence on AP sites for cccDNA degradation was confirmed by reduced intact DNA amount when DNA extracted from IFN-γ or TNF-α treated cells were digested with recombinant APE1, an AP endonuclease which specifically recognizes and cleaves AP sites. Surprisingly, knockdown of APE1 in IFN-γ and TNF-α treated HBV infected cells did not show a reduction in cccDNA clearance, suggesting the redundancy of endonucleases in clearing cccDNA. While AP sites can be repaired by the host cells’ DNA repair machinery, this is not observed with IFN-mediated AP site formation in cccDNA. This is most likely due to the concurrent downregulation of the base excision repair enzymes such as thymine DNA glycosylase and Nei-like DNA glycosylase after IFN treatment. These studies indicate that A3A and A3B are key proteins responsible for IFN and LTβR mediated cccDNA clearance. APOBECs also have other important roles in clearing HBV cccDNA. APOBEC3F (A3F) and APOBEC3G (A3G) were also found to have anti-HBV properties. A3G for example inhibits pgRNA packaging to reduce virion formation. The mechanism of action for A3F has not been well-characterized.
Figure 2 Interferon treatment silences covalently closed circular DNA transcription and recruits APOBECs to actively degrade covalently closed circular DNA.
Covalently closed circular DNA (cccDNA) exists as an episomal mini chromosome that is epigenetically modified to support active transcription. Interferon (IFN)-α treatment results in the recruitment of histone modifying complexes that remove activating transcription post-translational modifications (PTMs), and add repressive PTMs that silence cccDNA function. These complexes can be recruited by IFN-stimulated genes (ISGs) such as IFI16. ISGs may also bind directly to cccDNA to repress transcription. Other ISGs such as APOBECs induced by IFN signalling are also recruited by HBc, generating AP sites which lead to cccDNA degradation. HBV: Hepatitis B virus; IFN: Interferon; IRF: Interferon regulatory factor; JAK: Activate janus kinase; STAT: Signal transducers and activators of transcription; ISGs: Interferon-stimulated genes; ISRE: Interferon-stimulated response elements; cccDNA: Covalently closed circular DNA.
Epigenetic silencing and transcriptional repression
Apart from eliminating cccDNA, permanently silencing cccDNA is another strategy for the development of anti-HBV therapy. Several studies have shown that IFNs control the epigenetic silencing of cccDNA[84,85], spurring growing interest in controlling cccDNA transcriptional activity through epigenetic modifications. HBV cccDNA exists as episomal chromatin wound around cellular histones that undergo post-translational modifications (PTMs), altering cccDNA chromatin compaction hence accessibility to transcription regulators (Figure 2). Some of the histone marks associated with active cccDNA transcription include H3K4me3, H3K27ac, H3K122ac, and repressive PTMs include H3K27me3. Of note, the distribution of these histone marks varies across different types of HBV-producing samples. In the HepG2-NTCP cell model, histone PTM enrichment is mainly confined to the pre-core/core promoter (CP) region whereas in PHH, they are found throughout the genome with the greatest modifications at the X and pre-S1 promoter regions. In contrast, HBV-infected liver tissues have few PTMs at the CP but accumulate them near pre-S2 and the X promoter regions. The reason and basis for this variability is at present unclear.
cccDNA transcription activity is heavily influenced by the state of epigenetic modification by host cellular factors and PTMs. The cccDNA mini chromosome is heavily modified by various activating PTMs under normal circumstances (Figure 2). H3K4 methyltransferase Set domain containing 1A (Set1A) is recruited to cccDNA promoter sites by HBx, depositing the activating PTM H3K4me3 to drive active transcription. By modulating the expression of Set1A, the relative expression of H3K4me3 can be fine-tuned. IFN treatment disrupts cccDNA transcription activity by downregulating these epigenetic PTMs that support transcription. In studies using PHH, IFN-α specifically reduced trimethylation of H3K4 and acetylation of H3K27 and H3K122 on cccDNA chromatin to inhibit transcription of HBV RNA, but had negligible effect on epigenetic modification for the control promoters of ACTB and Nanog in the host genome.
IFNs also result in cccDNA transcriptional repression through active recruitment of complexes that confer transcription inhibitory epigenetic modifications. The repressive H3K27me3 PTM is induced by IFN-α through increased binding of polycomb repressive complex 2 (PRC2) to cccDNA. The importance of H3K27me3 in inhibiting cccDNA transcription was also confirmed in a separate study showing that upregulation of DNA methyltransferase 3a hypermethylates HBV cccDNA to repress transcription. IFNs have also been recently shown to inhibit succinylation of cccDNA histones, adding on to the transcriptionally repressed state brought about by hypoacetylation and/or methylation. This involves the succinylation of H3K79 by GCN5 histone succinyltransferase (also known as lysine acetyltransferase 2A), which corresponds to higher HBV replication. They further showed that low levels of succinylated H3K79 from GCN5-specific knockdown resulted in significantly reduced cccDNA levels, and that expression of GCN5 and cccDNA correlate well in HBV-infected individuals. More importantly, IFN-α treatment could overcome the effects of overexpressed GCN5 to reduce the levels of succinylated cccDNA, indicating that IFNs act upstream to control GCN5 function and repress cccDNA transcription, the specific mechanism of which remains to be elucidated. Taken together, IFNs epigenetically silence cccDNA function through the recruitment of epigenetic factor complexes that add repressive PTMs or remove activating PTMs so that cccDNA enters a transcriptionally repressed chromatin structural state. Importantly, IFNs can also do so by inducing ISGs that bring about both the removal of activating PTMs and the addition of suppressive PTMs. For example, IFI16 reduces cccDNA transcription activity by recruiting histone deacetylase 1 (HDAC1) and Sirtuin 1 (SIRT1) to increase repressive H3K27me3 PTMs on cccDNA, and concurrently impairs the recruitment of p300/CBP to prevent the addition of activating PTMs on cccDNA.
Interestingly, cccDNA also carries the ISRE cis-element, and this is critical for establishing IFN-mediated epigenetic changes on cccDNA. The HBV ISRE is located at the enhancer I/X promoter region[92,93], and is recognized by the ISGF3 complex and the ISGs IRF1 and IRF7. In transcriptionally active cccDNA, the HBV ISRE is bound by phosphorylated and unphosphorylated STAT1 and STAT2 transcription factors to active cccDNA (Figure 2). IFN treatment induces redistribution of the STAT proteins from the HBV ISRE towards the IFN signalling pathway, resulting in antiviral effects against HBV cccDNA by the upregulation of cccDNA-targeting ISGs. One of these ISGs is IRF9, which binds directly to the HBV ISRE element to suppress cccDNA transcription. When the HBV ISRE is mutated, loss of IRF9 binding was shown to abrogate IFN-induced suppression of cccDNA transcription. This is clinically significant, as mutations in the HBV ISRE affects CHB patient response to IFN treatment to render IFN treatment less effective. In addition, the HBV ISRE sequence is HBV genotype dependent, thus its sequence-dependent functionality partially accounts for differences in patient responder rates between carriers of HBV genotypes B and C. Another ISG, ISG20, also directly inhibits transcription from cccDNA by direct binding to the enhancer II/CP region. Higher ISG20 expression level also correlates to better response to IFN-α treatment in CHB patients and viral clearance in HBV-infected chimpanzees. With the withdrawal of activating transcription factors from cccDNA and binding of specific transcription repressors induced by IFN treatment, cccDNA transcription is further suppressed by epigenetic modifications of histones such as histone hypoacetylation which occurs through the recruitment of the HDAC1 and SIRT1, and hypermethylated by the PRC2 complex. Thus, in addition to epigenetic silencing of cccDNA, IFNs also suppress cccDNA transcription activity by generating IFN-induced transcription repressors specific to cccDNA.
Inhibition of cccDNA synthesis
IFNs are also known to directly inhibit cccDNA synthesis. The antiviral ISG myxovirus resistance protein 2 has been shown to reduce cccDNA formation when overexpressed. Its specific knockdown abrogates the loss of cccDNA induced by IFN-α, providing confirmation for its role in IFN-α induced reduction in cccDNA levels. It has been proposed that this occurs through inhibiting cccDNA synthesis from rcDNA, as well as from downregulated HBV transcripts.