scholarly journals Small-Molecule Effectors of Hepatitis B Virus Capsid Assembly Give Insight into Virus Life Cycle

2008 ◽  
Vol 82 (20) ◽  
pp. 10262-10270 ◽  
Author(s):  
Christina Bourne ◽  
Sejin Lee ◽  
Bollu Venkataiah ◽  
Angela Lee ◽  
Brent Korba ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Small Molecule ◽  
Self Assembly ◽  
Binding Pocket ◽  
Hbv Replication ◽  
B Virus ◽  
In Vitro Assembly ◽  
Kinetics Of

ABSTRACT The relationship between the physical chemistry and biology of self-assembly is poorly understood, but it will be critical to quantitatively understand infection and for the design of antivirals that target virus genesis. Here we take advantage of heteroaryldihydropyrimidines (HAPs), which affect hepatitis B virus (HBV) assembly, to gain insight and correlate in vitro assembly with HBV replication in culture. Based on a low-resolution crystal structure of a capsid-HAP complex, a closely related series of HAPs were designed and synthesized. These differentially strengthen the association between neighboring capsid proteins, alter the kinetics of assembly, and give rise to aberrant structures incompatible with a functional capsid. The chemical nature of the HAP variants correlated well with the structure of the HAP binding pocket. The thermodynamics and kinetics of in vitro assembly had strong and predictable effects on product morphology. However, only the kinetics of in vitro assembly had a strong correlation with inhibition of HBV replication in HepG2.2.15 cells; there was at best a weak correlation between assembly thermodynamics and replication. The correlation between assembly kinetics and virus suppression implies a competition between successful assembly and misassembly, small molecule induced or otherwise. This is a predictive and testable model for the mechanism of action of assembly effectors.

scholarly journals Phosphorothioate Di- and Trinucleotides as a Novel Class of Anti-Hepatitis B Virus Agents

2004 ◽  
Vol 48 (6) ◽  
pp. 2199-2205 ◽  
Author(s):  
Radhakrishnan P. Iyer ◽  
Yi Jin ◽  
Arlene Roland ◽  
John D. Morrey ◽  
Samir Mounir ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleoside Analogs ◽  
Hbv Dna ◽  
Parallel Synthesis ◽  
Hbv Replication ◽  
Long Term Treatment ◽  
B Virus ◽  
Dna Strand

ABSTRACT Several nucleoside analogs are under clinical development for use against hepatitis B virus (HBV). Lamivudine (3TC), a nucleoside analog, and adefovir dipivoxil (ADV), an acyclonucleotide analog, are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new antiviral agents with novel mechanisms of action. A library of more than 600 di- and trinucleotide compounds synthesized by parallel synthesis using a combinatorial strategy was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2.2.15. Through an iterative process of synthesis, lead optimization, and screening, three analogs were identified as potent inhibitors of HBV replication: dinucleotides ORI-7246 (drug concentration at which a 10-fold reduction of HBV DNA was observed [EC90], 1.4 μM) and ORI-9020 (EC90, 1.2 μM) and trinucleotide ORI-7170 (EC90, 7.2 μM). These analogs inhibited the replication of both strands of HBV DNA. No suppression of HBV protein synthesis or intracellular core particle formation by these analogs was observed. No inhibition of HBV DNA strand elongation by the analogs or their 5′-triphosphate versions was apparent in in vitro polymerase assays. Although the exact mechanism of action is not yet identified, present data are consistent with an inhibition of the HBV reverse transcriptase-directed priming step prior to elongation of the first viral DNA strand. In transient-transfection assays, these analogs inhibited the replication of 3TC-resistant HBV. Synergistic interactions in combination treatments between the analogs and either 3TC or ADV were observed. These compounds represent a novel class of anti-HBV molecules and warrant further investigation as potential therapeutic agents.

scholarly journals Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro

2007 ◽  
Vol 88 (12) ◽  
pp. 3270-3274 ◽  
Author(s):  
Marianne Bonvin ◽  
Jobst Greeve
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Point Mutations ◽  
Alternative Mechanism ◽  
Amino Terminal ◽  
Hbv Replication ◽  
B Virus ◽  
Carboxy Terminal ◽  
Deaminase Domain

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

scholarly journals Analysis of the Cytosolic Domains of the Hepatitis B Virus Envelope Proteins for Their Function in Viral Particle Assembly and Infectivity

2006 ◽  
Vol 80 (24) ◽  
pp. 11935-11945 ◽  
Author(s):  
Matthieu Blanchet ◽  
Camille Sureau
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Self Assembly ◽  
Inner Core ◽  
Envelope Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Virus Envelope ◽  
Particle Assembly ◽  
B Virus

ABSTRACT The hepatitis B virus (HBV) envelope proteins have the ability to assemble three types of viral particles, (i) the empty subviral particles (SVPs), (ii) the mature HBV virions, and (iii) the hepatitis delta virus (HDV) particles, in cells that are coinfected with HBV and HDV. To gain insight into the function of the HBV envelope proteins in morphogenesis of HBV or HDV virions, we have investigated subdomains of the envelope proteins that have been shown or predicted to lie at the cytosolic face of the endoplasmic reticulum membrane during synthesis, a position prone to interaction with the inner core structure. These domains, referred to here as cytosolic loops I and II (CYL-I and -II, respectively), were subjected to mutagenesis. The mutations were introduced in the three HBV envelope proteins, designated small, middle, and large (S-HBsAg, M-HBsAg, and L-HBsAg, respectively). The mutants were expressed in HuH-7 cells to evaluate their capacity for self-assembly and formation of HBV or HDV virions when HBV nucleocapsid or HDV ribonucleoprotein, respectively, was provided. We found that SVP-competent CYL-I mutations between positions 23 and 78 of the S domain were permissive to HBV or HDV virion assembly. One mutation (P29A) was permissive for synthesis of the S- and M-HBsAg but adversely affected the synthesis or stability of L-HBsAg, thereby preventing the assembly of HBV virions. Furthermore, using an in vitro infection assay based on the HepaRG cells and the HDV model, we have shown that particles coated with envelope proteins bearing CYL-I mutations were fully infectious, hence indicating the absence of an infectivity determinant in this region. Finally, we demonstrated that the tryptophan residues at positions 196, 199, and 201 in CYL-II, which were shown to exert a matrix function for assembly of HDV particles (I. Komla-Soukha and C. Sureau, J. Virol. 80:4648-4655, 2006), were dispensable for both assembly and infectivity of HBV virions.

scholarly journals Efficacies of Entecavir against Lamivudine-Resistant Hepatitis B Virus Replication and Recombinant Polymerases In Vitro

2002 ◽  
Vol 46 (8) ◽  
pp. 2525-2532 ◽  
Author(s):  
S. Levine ◽  
D. Hernandez ◽  
G. Yamanaka ◽  
S. Zhang ◽  
R. Rose ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cross Resistance ◽  
Wild Type ◽  
Resistance Mutations ◽  
Hbv Replication ◽  
B Virus ◽  
Inhibition Studies

ABSTRACT Entecavir (ETV) is a potent and selective inhibitor of hepatitis B virus (HBV) replication in vitro and in vivo that is currently in clinical trials for the treatment of chronic HBV infections. A major limitation of the current HBV antiviral therapy, lamivudine (3TC), is the emergence of drug-resistant HBV in a majority of treated patients due to specific mutations in the nucleotide binding site of HBV DNA polymerase (HBV Pol). To determine the effects of 3TC resistance mutations on inhibition by ETV triphosphate (ETV-TP), a series of in vitro studies were performed. The inhibition of wild-type and 3TC-resistant HBV Pol by ETV-TP was measured using recombinant HBV nucleocapsids, and compared to that of 3TC-TP. These enzyme inhibition studies demonstrated that ETV-TP is a highly potent inhibitor of wild-type HBV Pol and is 100- to 300-fold more potent than 3TC-TP against 3TC-resistant HBV Pol. Cell culture assays were used to gauge the potential for antiviral cross-resistance of 3TC-resistant mutants to ETV. Results demonstrated that ETV inhibited the replication of 3TC-resistant HBV, but 20- to 30-fold higher concentrations were required. To gain further perspective regarding the potential therapeutic use of ETV, its phosphorylation was examined in hepatoma cells treated with extracellular concentrations representative of drug levels in plasma in ETV-treated patients. At these concentrations, intracellular ETV-TP accumulated to levels expected to inhibit the enzyme activity of both wild-type and 3TC-resistant HBV Pol. These findings are predictive of potent antiviral activity of ETV against both wild-type and 3TC-resistant HBV.

scholarly journals In Vitro Characterization of the Anti-Hepatitis B Virus Activity and Cross-Resistance Profile of 2′,3′-Dideoxy-3′-Fluoroguanosine

2006 ◽  
Vol 50 (3) ◽  
pp. 955-961 ◽  
Author(s):  
A.-C. Jacquard ◽  
M.-N. Brunelle ◽  
C. Pichoud ◽  
D. Durantel ◽  
S. Carrouée-Durantel ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Activity ◽  
Drug Resistant ◽  
Free System ◽  
Hbv Replication ◽  
Duck Hepatitis ◽  
Huh7 Cells ◽  
B Virus

ABSTRACT The fluorinated guanosine analog 2′,3′-dideoxy-3′-fluoroguanosine (FLG) was shown to inhibit wild-type (wt) hepatitis B virus (HBV) replication in a human hepatoma cell line permanently expressing HBV. Experiments performed in the duck model of HBV infection also showed its in vivo antiviral activity. In this study, we investigated the mechanism of inhibition of FLG on HBV replication and its profile of antiviral activity against different HBV or duck hepatitis B virus (DHBV) drug-resistant mutants. We found that FLG-triphosphate inhibits weakly the priming of the reverse transcription compared to adefovir-diphosphate in a cell-free system assay allowing the expression of an enzymatically active DHBV reverse transcriptase. It inhibits more potently wt DHBV minus-strand DNA synthesis compared to lamivudine-triphosphate and shows a similar activity compared to adefovir-diphosphate. FLG-triphosphate was most likely a competitive inhibitor of dGTP incorporation and a DNA chain terminator. In Huh7 cells transiently transfected with different HBV constructs, FLG inhibited similarly the replication of wt, lamivudine-resistant, adefovir-resistant, and lamivudine-plus-adefovir-resistant HBV mutants. These results were consistent with those obtained in the DHBV polymerase assay using the same drug-resistant polymerase mutants. In conclusion, our data provide new insights in the mechanism of action of FLG-triphosphate on HBV replication and demonstrate its inhibitory activity on drug-resistant mutant reverse transcriptases in vitro. Furthermore, our results provide the rationale for further clinical evaluation of FLG in the treatment of drug-resistant virus infection and in the setting of combination therapy to prevent or delay drug resistance.

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 Strong and Selective Inhibitors of Hepatitis B Virus Replication among Novel N4-Hydroxy- and 5-Methyl-β-l-Deoxycytidine Analogues

2007 ◽  
Vol 51 (7) ◽  
pp. 2523-2530 ◽  
Author(s):  
E. Matthes ◽  
A. Funk ◽  
I. Krahn ◽  
K. Gaertner ◽  
M. von Janta-Lipinski ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Inhibitory Concentration ◽  
Hbv Dna ◽  
Selective Inhibitor ◽  
Effective Concentration ◽  
Active Inhibitor ◽  
Hbv Replication ◽  
B Virus

ABSTRACT Novel N4-hydroxy- and 5-methyl-modified β-l-deoxycytidine analogues were synthesized and evaluated as anti-hepatitis B virus (HBV) agents. Their in vitro efficiencies were investigated in HepG2.2.15 cells stably transfected with HBV. β-l-2′,3′-Didehydro-2′,3′-dideoxy-N4-hydroxycytidine (β-l-Hyd4C) was most effective in reducing secreted HBV DNA (50% effective concentration [EC50], 0.03 μM), followed by β-l-2′,3′-dideoxy-3′-thia-N4-hydroxycytidine (EC50, 0.51 μM), β-l-2′,3′-dideoxy-N4-hydroxycytidine (EC50, 0.55 μM), and β-l-5-methyl-2′-deoxycytidine (EC50, 0.9 μM). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the β-l-cytidine derivatives was also assessed. In accordance with the cell culture data, β-l-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 μM. The cytotoxicities of some of the 4-NHOH-modified β-l-nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH2 group. The 50% cytotoxic concentrations for β-l-Hyd4C in HepG2 and HL-60 cells were 2,500 μM and 3,500 μM, respectively. In summary, our results demonstrate that at least β-l-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.

scholarly journals Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus.

1997 ◽  
Vol 41 (7) ◽  
pp. 1444-1448 ◽  
Author(s):  
S F Innaimo ◽  
M Seifer ◽  
G S Bisacchi ◽  
D N Standring ◽  
R Zahler ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Line ◽  
Nucleoside Analogs ◽  
Hbv Replication ◽  
Mitochondrial Dna Content ◽  
B Virus ◽  
Antiviral Activities ◽  
Replicative Intermediates

BMS-200475 is a novel carbocyclic 2'-deoxyguanosine analog found to possess potent and selective anti-hepatitis B virus (anti-HBV) activity. BMS-200475 is distinguished from guanosine by replacement of the natural furanose oxygen on the sugar moiety with an exo carbon-carbon double bond. In the HepG2 stably transfected cell line 2.2.15, BMS-200475 had a 50% effective concentration (EC50) of 3.75 nM against HBV, as determined by analysis of secreted HBV DNA. Structurally related compounds with adenine, iodouracil, or thymine base substitutions were significantly less potent or were inactive. Direct comparison of the antiviral activities of BMS-200475 with those of a variety of other nucleoside analogs, including lamivudine (EC50 = 116.26 nM), demonstrated the clearly superior in vitro potency of BMS-200475 in 2.2.15 cells. Intracellular HBV replicative intermediates were uniformly reduced when cells were treated with BMS-200475, but rebounded after treatment was terminated. The concentration of BMS-200475 causing 50% cytotoxicity in 2.2.15 cell cultures was 30 microM, approximately 8,000-fold greater than the concentration required to inhibit HBV replication in the same cell line. Treatment with BMS-200475 resulted in no apparent inhibitory effects on mitochondrial DNA content.

scholarly journals Phenylpropenamide Derivatives AT-61 and AT-130 Inhibit Replication of Wild-Type and Lamivudine-Resistant Strains of Hepatitis B Virus In Vitro

2002 ◽  
Vol 46 (9) ◽  
pp. 3057-3060 ◽  
Author(s):  
William E. Delaney ◽  
Ros Edwards ◽  
Danni Colledge ◽  
Tim Shaw ◽  
Phil Furman ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleoside Analogue ◽  
Wild Type ◽  
Hbv Replication ◽  
B Virus ◽  
Resistant Strains

ABSTRACT The phenylpropenamide derivatives AT-61 and AT-130 are nonnucleoside analogue inhibitors of hepatitis B virus (HBV) replication. They inhibited the replication of wild-type HBV with 50% inhibitory concentrations of 21.2 ± 9.5 and 2.40 ± 0.92 μM, respectively, compared to 0.064 ± 0.020 μM lamivudine. There were no significant differences in sensitivity between wild-type and nucleoside analogue-resistant (rtL180M, rtM204I, and rtL180M + rtM204V) HBV.

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