scholarly journals Noncompetitive Inhibition of Hepatitis B Virus Reverse Transcriptase Protein Priming and DNA Synthesis by the Nucleoside Analog Clevudine

2013 ◽  
Vol 57 (9) ◽  
pp. 4181-4189 ◽  
Author(s):  
Scott A. Jones ◽  
Eisuke Murakami ◽  
William Delaney ◽  
Phillip Furman ◽  
Jianming Hu
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Viral Dna ◽  
Second Stage ◽  
Chronic Hepatitis B Virus ◽  
B Virus ◽  
Tenofovir Df ◽  
Protein Priming

ABSTRACTAll currently approved antiviral drugs for the treatment of chronic hepatitis B virus (HBV) infection are nucleos(t)ide reverse transcriptase inhibitors (NRTI), which inhibit the DNA synthesis activity of the HBV polymerase. The polymerase is a unique reverse transcriptase (RT) that has a novel protein priming activity in which HP initiates viral DNA synthesis using itself as a protein primer. We have determined the ability of NRTI-triphosphates (TP) to inhibit HBV protein priming and their mechanisms of action. While entecavir-TP (a dGTP analog) inhibited protein priming initiated specifically with dGTP, clevudine-TP (a TTP analog) was able to inhibit protein priming independently of the deoxynucleoside triphosphate (dNTP) substrate and without being incorporated into DNA. We next investigated the effect of NRTIs on the second stage of protein priming, wherein two dAMP nucleotides are added to the initial deoxyguanosine nucleotide. The obtained results indicated that clevudine-TP as well as tenofovir DF-DP strongly inhibited the second stage of protein priming. Tenofovir DF-DP was incorporated into the viral DNA primer, whereas clevudine-TP inhibited the second stage of priming without being incorporated. Finally, kinetic analyses using the HBV endogenous polymerase assay revealed that clevudine-TP inhibited DNA chain elongation by HP in a noncompetitive manner. Thus, clevudine-TP appears to have the unique ability to inhibit HBV RT via binding to and distorting the HP active site, sharing properties with both NRTIs and nonnucleoside RT inhibitors.

scholarly journals Dominant negative mutants of the duck hepatitis B virus core protein interfere with RNA pregenome packaging and viral DNA synthesis

Hepatology ◽  
1999 ◽  
Vol 30 (1) ◽  
pp. 308-315 ◽  
Author(s):  
Fritz von Weizsäcker ◽  
Josef Köck ◽  
Stefan Wieland ◽  
Wolf-Bernhard Offensperger ◽  
Hubert E. Blum
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Core Protein ◽  
Dominant Negative ◽  
Viral Dna ◽  
Duck Hepatitis B Virus ◽  
Virus Core ◽  
Duck Hepatitis ◽  
B Virus

scholarly journals Inhibition of hepatitis B virus by a novel L-nucleoside, 2'-fluoro-5-methyl-beta-L-arabinofuranosyl uracil.

1996 ◽  
Vol 40 (2) ◽  
pp. 380-386 ◽  
Author(s):  
S Balakrishna Pai ◽  
S H Liu ◽  
Y L Zhu ◽  
C K Chu ◽  
Y C Cheng
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Hepg2 Cells ◽  
Inhibitory Concentration ◽  
Viral Dna ◽  
B Virus ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

2'-Fluoro-5-methyl-beta-L-arabinofuranosyl uracil (L-FMAU) was discovered to have potent antiviral activity against hepatitis B virus (HBV). L-FMAU was more potent than its D-enantiomer and produced dose-dependent inhibition of the viral DNA replication in 2.2.15 cells (human HepG2 cells with the HBV genome), with a 50% inhibitory concentration of 0.1 microM. There was no inhibitory effect on HBV transcription or protein synthesis. In the 2.2.15 cell system, L-FMAU did not show any toxicity up to 200 microM, whereas the D-enantiomer was toxic, with a 50% inhibitory concentration of 50 microM. Repeated treatments of HepG2 cells with L-FMAU at a 1 microM concentration for 9 days did not result in any decrease in the total mitochondrial DNA content, suggesting that a mode of toxicity similar to that produced by 2',3'-dideoxycytidine is unlikely. Also at concentrations as high as 200 microM, L-FMAU did not adversely affect mitochondrial function as determined by lactic acid production by L-FMAU-treated hepatoma cells. L-FMAU was metabolized in the cells to its mono-, di-, and triphosphates, A dose-dependent inhibition of HBV DNA synthesis by L-FMAU triphosphate was observed in the DNA polymerase assays with isolated HBV particles, suggesting that the mode of action of this compound could involve viral polymerase. However, L-FMAU was not incorporated into the cellular DNA. Considering the potent inhibition of the viral DNA synthesis and the nontoxicity of L-FMAU towards the host DNA synthetic machinery, this compound should be further explored for development as asn anti-HBV drug.

scholarly journals In Vitro Reconstitution of a Functional Duck Hepatitis B Virus Reverse Transcriptase: Posttranslational Activation by Hsp90

2000 ◽  
Vol 74 (24) ◽  
pp. 11447-11455 ◽  
Author(s):  
Jianming Hu ◽  
Dana Anselmo
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Host Factors ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus ◽  
Protein Priming ◽  
Hsp90 Function

ABSTRACT Reverse transcription in hepatitis B viruses is initiated through a unique protein priming mechanism whereby the viral reverse transcriptase (RT) first assembles into a ribonucleoprotein (RNP) complex with its RNA template and then initiates DNA synthesis de novo using the RT itself as a protein primer. RNP formation and protein priming require the assistance of host cell factors, including the molecular chaperone heat shock protein 90 (Hsp90). To better understand the mechanism of RT activation by Hsp90, we have now mapped the minimal RT sequences of the duck hepatitis B virus that are required for chaperone binding, RNP formation, and protein priming. Furthermore, we have reconstituted in vitro both RNP formation and protein priming using purified RT proteins and host factors. Our results show that (i) Hsp90 recognizes two independent domains of the RT, both of which are necessary for RNP formation and protein priming; (ii) Hsp90 function is required not only to establish, but also to maintain, the RT in a state competent for RNA binding; and (iii) Hsp90 is not required during RT synthesis and can activate the RT posttranslationally. Based on these findings, we propose a model for Hsp90 function whereby the chaperone acts as an active interdomain bridge to bring the two RT domains into a poised but labile conformation competent for RNP formation. It is anticipated that the reconstitution system established here will facilitate the isolation of additional host factors required for RT functions and further elucidation of the mechanisms of RT activation.

scholarly journals Residues Arg703, Asp777, and Arg781 of the RNase H Domain of Hepatitis B Virus Polymerase Are Critical for Viral DNA Synthesis

2013 ◽  
Vol 88 (1) ◽  
pp. 154-163 ◽  
Author(s):  
C. Ko ◽  
Y.-C. Shin ◽  
W.-J. Park ◽  
S. Kim ◽  
J. Kim ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Rnase H ◽  
Viral Dna ◽  
B Virus
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