scholarly journals Synergistic Interactions between Hepatitis B Virus RNase H Antagonists and Other Inhibitors

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Elena Lomonosova ◽  
Adam Zlotnick ◽  
John E. Tavis
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Index Theorem ◽  
Rnase H ◽  
Mass Action ◽  
Significant Activity ◽  
B Virus ◽  
Mass Action Law

ABSTRACT Combination therapies are standard for management of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections; however, no such therapies are established for human hepatitis B virus (HBV). Recently, we identified several promising inhibitors of HBV RNase H (here simply RNase H) activity that have significant activity against viral replication in vitro. Here, we investigated the in vitro antiviral efficacy of combinations of two RNase H inhibitors with the current anti-HBV drug nucleoside analog lamivudine, with HAP12, an experimental core protein allosteric modulator, and with each other. Anti-HBV activities of the compounds were tested in a HepG2-derived cell line by monitoring intracellular core particle DNA levels, and cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The antiviral efficiencies of the drug combinations were evaluated using the median-effect equation derived from the mass-action law principle and combination index theorem of Chou and Talalay. We found that combinations of two RNase H inhibitors from different chemical classes were synergistic with lamivudine against HBV DNA synthesis. Significant synergism was also observed for the combination of the two RNase H inhibitors. Combinations of RNase H inhibitors with HAP12 had additive antiviral effects. Enhanced cytotoxicity was not observed in the combination experiments. Because of these synergistic and additive effects, the antiviral activity of combinations of RNase H inhibitors with drugs that act by two different mechanisms and with each other can be achieved by administering the compounds in combination at doses below the respective single drug doses.

Phosphorylation of hepatitis B virus core C-terminally truncated protein (Cp149) by PKC increases capsid assembly and stability

2008 ◽  
Vol 416 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Hang Kang ◽  
Jaehoon Yu ◽  
Guhung Jung
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Virus Titre ◽  
Capsid Assembly ◽  
Human Hepatoma Cells ◽  
Virus Core ◽  
C Terminus ◽  
B Virus

The HBV (hepatitis B virus) core is a phosphoprotein whose assembly, replication, encapsidation and localization are regulated by phosphorylation. It is known that PKC (protein kinase C) regulates pgRNA (pregenomic RNA) encapsidation by phosphorylation of the C-terminus of core, which is a component packaged into capsid. Neither the N-terminal residue phosphorylated by PKC nor the role of the C-terminal phosphorylation have been cleary defined. In the present study we found that HBV Cp149 (core protein C-terminally truncated at amino acid 149) expressed in Escherichia coli was phosphorylated by PKC at Ser106. PKC-mediated phosphorylation increased core affinity, as well as assembly and capsid stability. In vitro phosphorylation with core mutants (S26A, T70A, S106A and T114A) revealed that the Ser106 mutation inhibited phosphorylation of core by PKC. CD analysis also revealed that PKC-mediated phosphorylation stabilized the secondary structure of capsid. When either pCMV/FLAG-Cp149[WT (wild-type)] or pCMV/FLAG-S106A Cp149 was transfected into Huh7 human hepatoma cells, mutant capsid level was decreased by 2.06-fold with the S106A mutant when compared with WT, although the same level of total protein was expressed in both cases. In addition, when pUC1.2x and pUC1.2x/S106A were transfected, mutant virus titre was decreased 2.31-fold compared with WT virus titre. In conclusion, PKC-mediated phosphorylation increased capsid assembly, stability and structural stability.

Transbody against hepatitis B virus core protein inhibits hepatitis B virus replication in vitro

2015 ◽  
Vol 25 (2) ◽  
pp. 363-369 ◽  
Author(s):  
Yawen Wang ◽  
Yiping Li ◽  
Na Li ◽  
Qianqian Zhu ◽  
Lingyun Hui ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Virus Replication ◽  
Core Protein ◽  
Virus Core ◽  
B Virus

The pregenome/C RNA of duck hepatitis B virus is not used for translation of core protein during the early phase of infection in vitro

2015 ◽  
Vol 196 ◽  
pp. 13-19 ◽  
Author(s):  
Qiang Liu ◽  
Juan Huang ◽  
Renyong Jia ◽  
Mingshu Wang ◽  
Dekang Zhu ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Early Phase ◽  
Core Protein ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus

scholarly journals Properties of Monoclonal Antibodies Directed against Hepatitis B Virus Polymerase Protein

1999 ◽  
Vol 73 (5) ◽  
pp. 4188-4196 ◽  
Author(s):  
Jasper zu Putlitz ◽  
Robert E. Lanford ◽  
Rolf I. Carlson ◽  
Lena Notvall ◽  
Suzanne M. de la Monte ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Insect Cells ◽  
Rnase H ◽  
Hbv Replication ◽  
B Virus ◽  
Multifunctional Enzymes ◽  
Infected Insect

ABSTRACT Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.

scholarly journals Antiviral Properties and Mechanism of Action Studies of the Hepatitis B Virus Capsid Assembly Modulator JNJ-56136379

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Jan Martin Berke ◽  
Pascale Dehertogh ◽  
Karen Vergauwen ◽  
Wendy Mostmans ◽  
Koen Vandyck ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Hbv Dna ◽  
Size Exclusion ◽  
Capsid Assembly ◽  
Phase Ii Trials ◽  
B Virus

ABSTRACT Capsid assembly is a critical step in the hepatitis B virus (HBV) life cycle, mediated by the core protein. Core is a potential target for new antiviral therapies, the capsid assembly modulators (CAMs). JNJ-56136379 (JNJ-6379) is a novel and potent CAM currently in phase II trials. We evaluated the mechanisms of action (MOAs) and antiviral properties of JNJ-6379 in vitro. Size exclusion chromatography and electron microscopy studies demonstrated that JNJ-6379 induced the formation of morphologically intact viral capsids devoid of genomic material (primary MOA). JNJ-6379 accelerated the rate and extent of HBV capsid assembly in vitro. JNJ-6379 specifically and potently inhibited HBV replication; its median 50% effective concentration (EC50) was 54 nM (HepG2.117 cells). In HBV-infected primary human hepatocytes (PHHs), JNJ-6379, when added with the viral inoculum, dose-dependently reduced extracellular HBV DNA levels (median EC50 of 93 nM) and prevented covalently closed circular DNA (cccDNA) formation, leading to a dose-dependent reduction of intracellular HBV RNA levels (median EC50 of 876 nM) and reduced antigen levels (secondary MOA). Adding JNJ-6379 to PHHs 4 or 5 days postinfection reduced extracellular HBV DNA and did not prevent cccDNA formation. Time-of-addition PHH studies revealed that JNJ-6379 most likely interfered with postentry processes. Collectively, these data demonstrate that JNJ-6379 has dual MOAs in the early and late steps of the HBV life cycle, which is different from the MOA of nucleos(t)ide analogues. JNJ-6379 is in development for chronic hepatitis B treatment and may translate into higher HBV functional cure rates.

scholarly journals Inhibition of Hepatitis B Virus Polymerase by Entecavir

2007 ◽  
Vol 81 (8) ◽  
pp. 3992-4001 ◽  
Author(s):  
David R. Langley ◽  
Ann W. Walsh ◽  
Carl J. Baldick ◽  
Betsy J. Eggers ◽  
Ronald E. Rose ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Kinetic Studies ◽  
Hbv Dna ◽  
Cross Resistance ◽  
Significant Activity ◽  
B Virus ◽  
Dna Chain

ABSTRACT Entecavir (ETV; Baraclude) is a novel deoxyguanosine analog with activity against hepatitis B virus (HBV). ETV differs from the other nucleoside/tide reverse transcriptase inhibitors approved for HBV therapy, lamivudine (LVD) and adefovir (ADV), in several ways: ETV is >100-fold more potent against HBV in culture and, at concentrations below 1 μM, displays no significant activity against human immunodeficiency virus (HIV). Additionally, while LVD and ADV are obligate DNA chain terminators, ETV halts HBV DNA elongation after incorporating a few additional bases. Three-dimensional homology models of the catalytic center of the HBV reverse transcriptase (RT)-DNA-deoxynucleoside triphosphate (dNTP) complex, based on the HIV RT-DNA structure, were used with in vitro enzyme kinetic studies to examine the mechanism of action of ETV against HBV RT. A novel hydrophobic pocket in the rear of the RT dNTP binding site that accommodates the exocyclic alkene moiety of ETV was predicted, establishing a basis for the superior potency observed experimentally. HBV DNA chain termination by ETV was accomplished through disfavored energy requirements as well as steric constraints during subsequent nucleotide addition. Validation of the model was accomplished through modeling of LVD resistance substitutions, which caused an eightfold decrease in ETV susceptibility and were predicted to reduce, but not eliminate, the ETV-binding pocket, in agreement with experimental observations. ADV resistance changes did not affect the ETV docking model, also agreeing with experimental results. Overall, these studies explain the potency, mechanism, and cross-resistance profile of ETV against HBV and account for the successful treatment of naive and LVD- or ADV-experienced chronic HBV patients.

scholarly journals Interaction between Hepatitis B Virus Core Protein and Reverse Transcriptase

2000 ◽  
Vol 74 (24) ◽  
pp. 11479-11489 ◽  
Author(s):  
Lisa Lott ◽  
Burton Beames ◽  
Lena Notvall ◽  
Robert E. Lanford
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Binding Sites ◽  
Core Protein ◽  
Rnase H ◽  
Genome Replication ◽  
Stem Loop ◽  
B Virus ◽  
Carboxy Terminal

ABSTRACT Previous mutagenesis studies with hepatitis B virus (HBV) suggest that continued interactions with core are required for several steps in genomic replication. To examine core-polymerase (Pol) interactions, insect cells were coinfected with baculovirus constructs that independently expressed core and Pol. The results demonstrated several features with implications that core plays an interactive role with HBV Pol: (i) core coprecipitated with constructs expressing full-length Pol as well as the terminal protein (TP), reverse transcriptase (RT) and RNase H domains of Pol, independently; (ii) coprecipitation of core was not dependent on the presence of an epsilon stem-loop sequence; and (iii) core-Pol complexes migrated as intact capsid particles, as detected by sucrose gradient analysis. To analyze the structural and sequence requirements of core in recognition of Pol, a series of core mutants with two- to four-amino-acid insertions or carboxy-terminal deletions were assessed for Pol interaction. The results indicated that capsid formation is required but not sufficient for interaction with Pol and that the TP and RT domains of Pol have different requirements for interaction with core. To map the core binding sites on Pol, a panel of amino- and carboxy-terminal deletion mutants of the TP and RT domains of Pol were analyzed for interaction with core. At least three separate core binding sites on Pol were detected. This analysis begins to define basic requirements for core-Pol interactions, but further study is necessary to delineate the effects of these interactions on encapsidation and genome replication.

scholarly journals Intracellular Distribution of Hepatitis B Virus Core Protein Expressed In Vitro Depends on the Sequence of the Isolate and the Serologic Pattern

2004 ◽  
Vol 189 (9) ◽  
pp. 1634-1645 ◽  
Author(s):  
Mohammed S. Jazayeri ◽  
Edward S. Dornan ◽  
Winifred Boner ◽  
Giovanna Fattovich ◽  
Stephanos Hadziyannis ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Intracellular Distribution ◽  
Virus Core ◽  
B Virus

scholarly journals Anti-Hepatitis B Virus Effect and Possible Mechanism of Action of 3,4-O-Dicaffeoylquinic AcidIn VitroandIn Vivo

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Yi-Hang Wu ◽  
Bing-Jie Hao ◽  
Hong-Cui Cao ◽  
Wei Xu ◽  
Yong-Jun Li ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Transgenic Mice ◽  
Core Protein ◽  
Heme Oxygenase 1 ◽  
Test Compound ◽  
Hbv Cccdna ◽  
Dicaffeoylquinic Acid ◽  
B Virus

The anti-hepatitis B activity of 3,4-O-dicaffeoylquinic acid isolated fromLaggera alatawas studied using theD-galactosamine- (D-GalN-) induced hepatocyte damage model, HepG2.2.15 cells, and with HBV transgenic mice.In vitroresults showed that 3,4-O-dicaffeoylquinic acid improved HL-7702 hepatocyte viability and markedly inhibited the production of HBsAg and HBeAg. At a concentration of 100 μg/mL, its inhibitory rates on the expression levels of HBsAg and HBeAg were 89.96% and 81.01%, respectively. The content of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in HepG2.2.15 cells was significantly decreased after the cells were treated with the test compound. In addition, 3,4-O-dicaffeoylquinic acid significantly increased the expression of heme oxygenase-1 (HO-1) in HepG2.2.15 cells.In vivoresults indicated that the test compound at concentrations of 100 μg/mL significantly inhibited HBsAg production and increased HO-1 expression in HBV transgenic mice. In conclusion, this study verifies the anti-hepatitis B activity of 3,4-O-dicaffeoylquinic acid. The upregulation of HO-1 may contribute to the anti-HBV effect of this compound by reducing the stability of the HBV core protein, which blocks the refill of nuclear HBV cccDNA. Furthermore, the hepatoprotective effect of this compound may be mediated through its antioxidative/anti-inflammatory properties and by the induction of HO-1 expression.

scholarly journals Full-Length Hepatitis B Virus Core Protein Packages Viral and Heterologous RNA with Similarly High Levels of Cooperativity

2010 ◽  
Vol 84 (14) ◽  
pp. 7174-7184 ◽  
Author(s):  
J. Zachary Porterfield ◽  
Mary Savari Dhason ◽  
Daniel D. Loeb ◽  
Michael Nassal ◽  
Stephen J. Stray ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Binding ◽  
Core Protein ◽  
Viral Polymerase ◽  
Critical Feature ◽  
Protein Dimer ◽  
B Virus

ABSTRACT A critical feature of a viral life cycle is the ability to selectively package the viral genome. In vivo, phosphorylated hepatitis B virus (HBV) core protein specifically encapsidates a complex of pregenomic RNA (pgRNA) and viral polymerase; it has been suggested that packaging is specific for the complex. Here, we test the hypothesis that core protein has intrinsic specificity for pgRNA, independent of the polymerase. For these studies, we also evaluated the effect of core protein phosphorylation on assembly and RNA binding, using phosphorylated core protein and a phosphorylation mimic in which S155, S162, and S170 were mutated to glutamic acid. We have developed an in vitro system where capsids are disassembled and assembly-active core protein dimer is purified. With this protein, we have reassembled empty capsids and RNA-filled capsids. We found that core protein dimer bound and encapsidated both the HBV pregenomic RNA and heterologous RNA with high levels of cooperativity, irrespective of phosphorylation. In direct competition assays, no specificity for pregenomic RNA was observed. This suggests that another factor, such as the viral polymerase, is required for specific packaging. These results also beg the question of what prevents HBV core protein from assembling on nonviral RNA, preserving the protein for virus production.

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