Interferon Alpha Induces Multiple Cellular Proteins That Coordinately Suppress Hepadnaviral Covalently Closed Circular DNA Transcription.

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of an infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that downregulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. In contrast, ectopic expression of STAT1, SMCHD1, or PML significantly reduced cccDNA transcription activity. SMCHD1 is a noncanonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and is involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1, and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced posttranslational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1, and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-α is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant, fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual IFN-stimulated genes (ISGs) on hepadnaviral mRNAs transcribed from cccDNA, we found that downregulating the expression of STAT1, SMCHD1, or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the posttranslational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.

Developments in Cell-Penetrating Peptides as Antiviral Agents and as Vehicles for Delivery of Peptide Nucleic Acid Targeting Hepadnaviral Replication Pathway.

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.

Carbonyl J acid derivatives block protein priming of hepadnaviral P protein and DNA-dependent DNA synthesis activity of hepadnaviral nucleocapsids.

Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 µM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.

Alkylated porphyrins have broad antiviral activity against hepadnaviruses, flaviviruses, filoviruses, and arenaviruses.

We screened ∼2,200 compounds known to be safe in people for the ability to reduce the amount of virion-associated hepatitis B virus (HBV) DNA in the culture medium of producer cells. These efforts led to the discovery of an alkylated porphyrin, chlorophyllide, as the compound that achieved the greatest reduction in signal. Here we report that chlorophyllide directly and quantitatively disrupted HBV virions at micromolar concentrations, resulting in the loss of all detectable virion DNA, without detectably affecting cell viability or intracellular viral gene products. Chemophores of chlorophyllide were also tested. Chlorin e6, a metal-free chlorophyllide-like molecule, showed the strongest antiviral activity against HBV as well as profound antiviral effects on other enveloped viruses, such as hepatitis C virus (HCV), human immunodeficiency virus (HIV), dengue virus (DENV), Marburg virus (MARV), Tacaribe virus (TCRV), and Junin viruses (JUNV). Remarkably, chlorin e6 inactivated DENV at subnanomolar-level concentrations. However, the compound had no antiviral effect against encephalomyocarditis virus and adenovirus, suggesting that chlorin e6 may be less active or inactive against nonenveloped viruses. Although other porphyrin derivatives have been previously reported to possess antiviral activity, this is the first analysis of the biochemical impact of chlorophyllide and chlorin e6 against HBV and of the dramatic anti-infectivity impact upon DENV. The possible application of this family of compounds as antiviral agents, as microbicides and systemic virus neutralizing agents, is discussed.

Antiviral Chemistry & Chemotherapy's current antiviral agents FactFile (2nd edition): retroviruses and hepadnaviruses.

There are at present exactly 25 compounds that have been formally approved for the treatment of retrovirus (that is HIV) infections: seven nucleoside reverse transcriptase inhibitors (NRTIs), one nucleotide reverse transcriptase inhibitor (NtRTI), four non-nucleoside reverse transcriptase inhibitors (NNRTIs), 10 protease inhibitors (PIs), one coreceptor inhibitor (CRI), one fusion inhibitor (FI) and one integrase inhibitor (INI). Other compounds expected to be approved for the treatment of HIV infections in the near future are the NNRTI rilpivirine, the CRI vicriviroc and the INI elvitegravir. To obtain synergistic activity, enable lower dosage levels, thus minimizing toxic side effects, and particularly to reduce the risk of drug resistance development, common wisdom dictates that the HIV inhibitors should be used in drug combination regimens. Although, given the number of compounds available, the drug combinations that could be concocted are uncountable, only one triple-drug combination has so far been formulated as single pill to be taken orally once daily, namely Atripla containing the NtRTI tenofovir disoproxil fumarate, the NRTI emtricitabine and the NNRTI efavirenz. Here, we document these approved compounds along with other HIV-active compounds and, for the first time, compounds whose principal activity is against hepatitis B virus. The logic of this new division being the enzymatic similarity between the reverse transcriptase of HIV and hepatitis B virus; the strategies for the development of antiviral agents to combat them have much in common.

Cross-resistance testing of antihepadnaviral compounds using novel recombinant baculoviruses which encode drug-resistant strains of hepatitis B virus.

Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in the selection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistant variants are clearly warranted but have been difficult to do because of the lack of convenient and reliable in vitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replication that relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBV genome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently been used to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation. The further development of this novel system and its application to determine the cross-resistance profiles of drug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses which encoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examine the effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir), and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526M mutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutations M550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir is active against each of these mutants. These findings are supported by the limited amount of clinical data currently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecular characterization of clinically significant HBV strains.

Impact of viral oncogenesis on responses to anti-cancer drugs and irradiation.

The view that chemical or physical oncogenesis and tumor therapy resistance represent different parts of common cellular alterations gained considerable attractiveness, because it explains the inherent unreponsiveness of many tumors. Viruses are potent oncogenes and are causally linked to approximately one-fifth of all human malignancies. Whether viral oncogenesis exerts comparable effects was less clear. Recent progress in experimental research provided ample evidence that viruses affect response of tumor cells toward anti-cancer drugs and irradiation. Resistance to cytostatic drugs and radiation develops by alterations at the drug-target sites (i.e., DNA or specific target proteins), upstream (i.e., detoxification mechanisms), or downstream of them (i.e., programmed cell death). Viruses interfere with specific cellular genes at these three levels. Viral proteins induce the expression and expression of drug resistance genes, that is, MDR1, DHFR, or CAD. Viral interactions with the tumor suppressor genes (p53, pRB) abrogate cell cycle arrests and disturb DNA repair of drug- and radiation-induced DNA lesions. The readiness to commit cellular suicide (apoptosis) is also affected by viral genes. The connection between viral oncogenesis and the response of tumor cells to treatment adds a new dimension to tumor biology and may have important consequences for oncological treatment modalities in the future.

The problem of antiviral therapy for chronic hepadnavirus infections.

Attempts at antiviral therapy of patients with active liver disease as a consequence of chronic hepatitis B virus infection have been moderately successful. The molecular and cellular basis for a successful outcome in these patients is not understood and the same therapies do not appear to benefit carriers that still have fairly normal livers and only a moderate hepatitis as a result of the immune response to the infection. Most carriers fall into this latter classification, at least during the early years of infection, and a therapy that could be successfully applied before extensive liver damage had occurred would presumably reduce the risk of subsequent liver damage and the progression to primary hepatocellular carcinoma. Traditionally, it has been assumed that the primary reason that individuals become chronically infected is that the cytotoxic T-cell response and/or antibody-dependent killing of infected hepatocytes is insufficient to clear the infection. Less attention has been focused on the role of the antibody response in the generation of virus-neutralizing antibodies as the possible major deficiency predisposing some individuals to become carriers. However, carriers normally are antigenemic for HBsAg and virus, and carriers with only antibodies to these structures in their circulation are virtually unknown. In addition, it is usually assumed that the hepatocyte, the major target of infection, does not spontaneously turn over and that, in the absence of an immune response to the infected cell, hepatocellular viability is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunosuppression with cyclosporine during the incubation period of experimental woodchuck hepatitis virus infection increases the frequency of chronic infection in adult woodchucks.

The immunosuppressive agent cyclosporine was given to adult woodchucks during acute experimental infection with woodchuck hepatitis virus (WHV). All 17 woodchucks given WHV alone or with a vehicle resolved the infection (i.e., zero chronicity), but when cyclosporine was given throughout the incubation and acute phases of infection (0-12 or 14 weeks; n = 12), the rate of chronic infection increased to 92%. When cyclosporine was given only during the incubation period (0-4 weeks; n = 10) or only during the acute phase of infection (2-12 weeks; n = 9), the rates increased to 50% and 55%, respectively. However, when the drug was given after the acute phase (8-18 weeks; n = 9), the chronic infection rate (11%) did not differ from that in untreated and vehicle controls. Immune responses inhibited by cyclosporine are important in resolution of acute WHV infection and occur mainly during the first 8 weeks. Immunosuppression of these responses for even short intervals during incubation (e.g., 0-4 weeks) increases the risk of chronicity.

Adenine arabinoside monophosphate and acyclovir monophosphate coupled to lactosaminated albumin reduce woodchuck hepatitis virus viremia at doses lower than do the unconjugated drugs.

The woodchuck was selected to study the efficacy of liver-targeted antiviral drugs on hepadnavirus replication. Nineteen woodchucks chronically infected with woodchuck hepatitis virus were treated with adenine arabinoside monophosphate or acyclovir monophosphate, either free or conjugated with the liver-targeting molecule lactosaminated human serum albumin. Circulating woodchuck hepatitis virus DNA levels remained unchanged in untreated animals and in those receiving the carrier lactosaminated human serum albumin alone; in contrast, they were consistently lower after 5 days of treatment with the antiviral drugs. Free and conjugated adenine arabinoside monophosphate were active at doses of 10 and 0.75 mg/kg, respectively, and free and coupled ACVMP were active at doses of 20 and 2.6 mg/kg, respectively. These results indicate that the dosages of adenine arabinoside monophosphate and acyclovir monophosphate required to inhibit hepadnavirus growth can be sharply reduced by coupling the drugs to lactosaminated human serum albumin.

Hepadnavirus enhancer and its binding proteins.

In the hepadnavirus enhancer region, a 33 bp DNA sequence is strongly conserved among mammalian hepadnavirus genomes. To elucidate the role of the sequence, we tested enhancer activities and capability to form DNA-protein complex of several synthetic DNAs. Not only two tandem copies of a 46 bp DNA covering the sequence but also two tandem copies of a 23 bp in the sequence exhibit enhancer activity. Also the activity was augmented by treatment of a tumor promoter, TPA. DNA binding proteins complexes with the 23 bp DNA were augmented in extracts of HepG2 or HeLa cells stimulated with TPA. These results imply that the conserved sequence of hepadnavirus enhancer is a TPA-inducible enhancer which is transactivated by ubiquitous DNA-binding proteins. We presented results showing that DNA-protein complexes with a 23 bp DNA are similar to but distinct from those with a TPA-responsive element DNA, the recognition site for c-jun/fos products. We also presented results suggesting that hepadnavirus X protein may not directly or indirectly affect DNA-protein complex formation with the conserved sequence in the hepadnavirus enhancer.

Inhibitory effects of 2'-fluorinated arabinosyl-pyrimidine nucleosides on woodchuck hepatitis virus replication in chronically infected woodchucks.

The treatment of woodchuck hepatitis virus infections with 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), given intraperitoneally, caused complete and permanent decrease of serum virus endogenous DNA polymerase and viral DNA in all treated woodchucks but was associated with severe toxicity. By contrast 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-ethyluracil (FEAU) induced a sustained, although less dramatic, decrease of viral replication without apparent toxic effect. FEAU was also effective when given orally. However, in both cases this inhibitory effect was transient.