Regulation of viral expression by the HBV core protein and the characterization HBc as a potential therapeutic target for HBV cure

Background and Aim The hepatitis B virus (HBV) is a DNA virus organized into nucleosomal structures. HBV produces covalently closed double-stranded DNA (cccDNA) that is found in the nuclei of infected cells as a viral minichromosome. HBV encoded proteins HBx and HBc both bind the cccDNA early after infection. HBx is required for the inactivation of the HBV cellular restriction factor smc5/6 and to establish and maintain cccDNA transcription. HBc binding to the cccDNA in thought to contribute to cccDNA chromatinization and nucleosome spacing whereas its impact on cccDNA transcription is still debated. Increasing evidence indicate that HBV proteins regulating viral minichromosomes also interact with the cellular chromatin and chromatin modifying enzymes to target cellular genes expression through epigenetic modifications. A ChIP-Seq genome wide analysis of HBx chromatin recruitment in HBV replicating cells has defined a broad repertoire cellular genes (~4.000) of and ncRNAs (75 miRNAs and 34 lncRNAs) potentially regulated by HBx with an enrichment in genes/ncRNAs involved in cell metabolism, chromatin dynamics and cancer but also in genes/ncRNAs that modulate HBV replication. HBc has also been shown to bind to cellular chromatin (~1000 genes) and to the promoters of a subset of cellular genes involved in inflammatory responses and innate immunity. Core protein (Cp) represents an attractive new therapeutic specific target for HBV chronic infection. Cp is essential for HBV genome packaging, reverse transcription, intracellular trafficking and the re-import of encapsidated HBV genomes into the nucleus, but due to its nuclear functions drugs targeting the Cp may also impact on cccDNA transcriptional activity and host genes reprogramming with a great potential for enhanced antiviral activity. The elucidation of the HBc crystal structure and the clarification of core dimers assembly process has led to the development of several compounds that target Cp and capsid assembly inhibit HBV replication therapeutic potential. We focused on two different classes of Cp assembly modulators. Heteroaryldihydropyrimidines (HAPs) act as allosteric effectors to increase the kinetics of assembly, strengthen dimer-dimer association and prevent the proper formation of viral capsids with the formation of aberrant core particles at high concentrations. The phenyl-propenamide derivatives AT-61 and AT-130 increase capsid assembly reaction rate, interfere with HBV RNA packaging and to the formation of apparently normal empty capsids. Interestingly, compounds belonging to the HAP chemical class have moved into clinical trials. In this third year of my PhD I further investigated the interplay between the impact of HAP12 and AT-130 treatment on viral replication and HBV core recruitment on the cccDNA and I expanded these observation on the regulation of cellular genes targeted by Cp. Methods I made use of three cellular models of HBV infection/replication: a) HepAD38 cells, a stable clone derived from the hepatocellular cell line HepG2, carry a complete integrated HBV genome under the control of a tetracycline- off inducible promoter; a HepG2-NTCP stable clone expressing high levels of the HBV entry receptor Na+/taurocholate co-transporting polypeptide (NTCP), that allow HBV infection in vitro; primary human hepatocytes (PHHs) from multiple donors infeted with HBV inocula purified from the cell supernatants of HepAD38 stable cell lines. Cells were treated with the anti-capsid HAP12 and AT-130 respectively 1 and 5 μM concentration to assess the effects on viral replication. All relevant virological parameters were evaluated: capsid-associated HBV-DNA (TaqMan real-time PCR); cccDNA levels (TaqMan real-time PCR); pgRNA levels (quantitative real-time PCR). Nuclear cccDNA was visualized by DNA FISH using using HBV specific probes encompassing the whole HBV genome. Anti-HBc, anti-HBx and anti-AcH4 ChIPs and cccDNA-ChIP experiments were performed in TET-released HepAD38 cells and in mock and HBV-infected NTCP-HepG2 cells and PHHs and analyzed by TaqMan real-time PCR using cccDNA and gene specific primers. Results I have shown that HAP12 and AT130, in addition to suppress efficiently HBV replication prevents the accumulation of nuclear cccDNA by blocking the recycling of mature core particles into the nucleus in TET released HepAD38 cells. Similarly, HAP12 treatment of NTCP-HepG2 cells at the time of HBV infection results in a drastic reduction of cccDNA formation, suggesting an important role of Cp for rcDNA release into the nucleus, conversion of rcDNA into cccDNA or cccDNA chromatinization. Conversely, when HAP12 treatment was started 10 days post-infection, when the cccDNA pool is established and stable in HBV infected NTCP-HepG2 cells, I observed very little or no effect on cccDNA levels, a significant reduction of cccDNA transcription and pgRNA levels together with a very strong inhibition of total HBV DNA and viral replication. These results indicate that HAP12, in addition to target capsid formation and pgRNA encapsidation in the cytoplasm, also affect cccDNA function in the nucleus. Next, I confirmed that Cp binds to the cccDNA in all the HBV infection/replication models and I showed that Cp recruitment on the cccDNA occurs as early as 2 hours post-infection in HBV infected PHHs, preceding the binding of HBx onto the cccDNA that becomes evident at 4-8 hours post-infection. Finally, I investigated the ability of Cp to bind to selected cellular promoters in HBV infected cells and the HAP12 onto Cp recruitment on these promoters. To this aim I performed anti-HBc ChIP assays in TET released HepAD38 cells, HBV infected NTCP-HepG2 cells and HBV infected PHHs and I confirmed Cp binding to the regulatory regions of the Ezh2 histon methyl-transferase, the cSrc proto-oncogene (that has been also shown to potentiate HBV replication), the E2F1 transcription factor and cell cycle regulator, and the IL29/lamda3 interferon, whereas the IL6 promoter was consitently not enriched and served as negative control. Importantly, HAP treatment was able to blocks Cp recruitment on genes promoters. Altogether these results identify capsid inhibitors as the first class of “virus specific” compounds capable to target the cccDNA functions and potentially counteract Cp pathogenicity in infected hepatocytes.