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Alternative therapeutic approaches against chronic hepatitis B virus (HBV) infection need to be urgently developed because current therapies are only virostatic. In this context, cell penetration peptides (CPPs) and their Peptide Nucleic Acids (PNAs) cargoes appear as a promising novel class of biologically active compounds. In this review we summarize different in vitro and in vivo studies, exploring the potential of CPPs as vehicles for intracellular delivery of PNAs targeting hepadnaviral replication. Thus, studies conducted in the duck HBV (DHBV) infection model showed that conjugation of (D-Arg)₈ CPP to PNA targeting viral epsilon (ε) were able to efficiently inhibit viral replication in vivo following intravenous administration to ducklings. Unexpectedly, some CPPs, (D-Arg)₈ and Decanoyl-(D-Arg)₈, alone displayed potent antiviral effect, altering late stages of DHBV and HBV morphogenesis. Such antiviral effects of CPPs may affect the sequence-specificity of CPP-PNA conjugates. By contrast, PNA conjugated to (D-Lys)₄ inhibited hepadnaviral replication without compromising sequence specificity. Interestingly, Lactose-modified CPP mediated the delivery of anti-HBV PNA to human hepatoma cells HepaRG, thus improving its antiviral activity. In light of these promising data, we believe that future studies will open new perspectives for translation of CPPs and CPP-PNA based technology to therapy of chronic hepatitis B.

Hepatitis delta virus (HDV) increases morbidity in Hepatitis B virus (HBV)-infected patients. In the mid-eighties, an outbreak of HDV fulminant hepatitis (FH) in the Central African Republic (CAR) killed 88% of patients hospitalized in Bangui. We evaluated infections with HBV and HDV among students and pregnant women, 25 years after the fulminant hepatitis (FH) outbreak to determine (i) the prevalence of HBV and HDV infection in this population, (ii) the clinical risk factors for HBV and/or HDV infections, and (iii) to characterize and compare the strains from the FH outbreak in the 1980s to the 2010 HBV-HDV strains. We performed a cross sectional study with historical comparison on FH-stored samples (n = 179) from 159 patients and dried blood-spots from volunteer students and pregnant women groups (n = 2172). We analyzed risk factors potentially associated with HBV and HDV. Previous HBV infection (presence of anti-HBc) occurred in 345/1290 students (26.7%) and 186/870 pregnant women (21.4%)(p = 0.005), including 110 students (8.8%) and 71 pregnant women (8.2%), who were also HBsAg-positive (p = 0.824). HDV infection occurred more frequently in pregnant women (n = 13; 18.8%) than students (n = 6; 5.4%) (p = 0.010). Infection in childhood was probably the main HBV risk factor. The risk factors for HDV infection were age (p = 0.040), transfusion (p = 0.039), and a tendency for tattooing (p = 0.055) and absence of condom use (p = 0.049). HBV-E and HDV-1 were highly prevalent during both the FH outbreak and the 2010 screening project. For historical samples, due to storage conditions and despite several attempts, we could only obtain partial HDV amplification representing 25% of the full-length genome. The HDV-1 mid-eighties FH-strains did not form a specific clade and were affiliated to two different HDV-1 African subgenotypes, one of which also includes the 2010 HDV-1 strains. In the Central African Republic, these findings indicate a high prevalence of previous and current HBV-E and HDV-1 infections both in the mid-eighties fulminant hepatitis outbreak and among asymptomatic young adults in 2010, and reinforce the need for universal HBV vaccination and the prevention of HDV transmission among HBsAg-positive patients through blood or sexual routes.

Hepatitis B virus (HBV) infection remains a major public health concern worldwide with 240 million individuals chronically infected and at risk of developing cirrhosis and hepatocellular carcinoma. Current treatments rarely cure chronic hepatitis B infection, highlighting the need for new anti-HBV drugs. Nucleic acid polymers (NAPs) are phosphorothioated oligonucleotides that have demonstrated a great potential to inhibit infection with several viruses. In chronically infected human patients, NAPs administration lead to a decline of blood HBsAg and HBV DNA and to HBsAg seroconversion, the expected signs of functional cure. NAPs have also been shown to prevent infection of duck hepatocytes with the Avihepadnavirus duck hepatitis B virus (DHBV) and to exert an antiviral activity against established DHBV infection in vitro and in vivo. In this study, we investigated the specific anti-HBV antiviral activity of NAPs in the HepaRG human hepatoma cell line and primary cultures of human hepatocytes. NAPs with different chemical features (phosphorothioation, 2'O-methyl ribose, 5-methylcytidine) were assessed for antiviral activity when provided at the time of HBV inoculation or post-inoculation. NAPs dose-dependently inhibited HBV entry in a phosphorothioation-dependent, sequence-independent and size-dependent manner. This inhibition of HBV entry by NAPs was impaired by 2'O-methyl ribose modification. NAP treatment after viral inoculation did not elicit any antiviral activity.

Chronic hepatitis B virus infection is a leading cause of cirrhosis and liver cancer. Hepatitis B virus encodes the regulatory HBx protein whose primary role is to promote transcription of the viral genome, which persists as an extrachromosomal DNA circle in infected cells. HBx accomplishes this task by an unusual mechanism, enhancing transcription only from extrachromosomal DNA templates. Here we show that HBx achieves this by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the 'structural maintenance of chromosomes' (Smc) complex Smc5/6 for degradation. Blocking this event inhibits the stimulatory effect of HBx both on extrachromosomal reporter genes and on hepatitis B virus transcription. Conversely, silencing the Smc5/6 complex enhances extrachromosomal reporter gene transcription in the absence of HBx, restores replication of an HBx-deficient hepatitis B virus, and rescues wild-type hepatitis B virus in a DDB1-knockdown background. The Smc5/6 complex associates with extrachromosomal reporters and the hepatitis B virus genome, suggesting a direct mechanism of transcriptional inhibition. These results uncover a novel role for the Smc5/6 complex as a restriction factor selectively blocking extrachromosomal DNA transcription. By destroying this complex, HBx relieves the inhibition to allow productive hepatitis B virus gene expression.

Background & Aims: Maintenance of the covalently closed circular HBV DNA (cccDNA) that serves as a template for HBV transcription is responsible for the failure of antiviral therapies. While studies in chronic hepatitis patients have shown that high viremia correlates with hyperacetylation of cccDNA-associated histones, the molecular mechanisms controlling cccDNA stability and transcriptional regulation are still poorly understood. This study aimed to decipher the role of chromatin and chromatin modifier proteins on HBV transcription., Methods: We analyzed the chromatin structure of actively transcribed or silenced cccDNA by infecting primary human hepatocytes and differentiated HepaRG cells with wild-type virus or virus deficient (HBVX-) for the expression of hepatitis B virus X protein (HBx), that is required for HBV expression., Results: In the absence of HBx, HBV cccDNA was transcriptionally silenced with the concomitant decrease of histone 3 (H3) acetylation and H3K4me3, increase of H3 di- and tri-methylation (H3K9me) and the recruitment of heterochromatin protein 1 factors (HP1) that correlate with condensed chromatin. SETDB1 was found to be the main histone methyltransferase responsible for the deposition of H3K9me3 and HBV repression. Finally, full transcriptional reactivation of HBVX- upon HBx re-expression correlated with an increase of histone acetylation and H3K4me3, and a concomitant decrease of HP1 binding and of H3K9me3 on the cccDNA., Conclusion: Upon HBV infection, cellular mechanisms involving SETDB1-mediated H3K9me3 and HP1 induce silencing of HBV cccDNA transcription through modulation of chromatin structure. HBx is able to relieve this repression and allow the establishment of active chromatin., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)