Genotyping the hepatitis B virus with a fragment of the HBV DNA polymerase gene in Shenyang, China

The hepatitis B virus (HBV) has been classified into eight genotypes (A-H) based on intergenotypic divergence of at least 8% in the complete nucleotide sequence or more than 4% in the S gene. To facilitate the investigation of the relationship between the efficacy of drug treatment and the mutation with specific genotype of HBV, we have established a new genotyping strategy based on a fragment of the HBV DNA polymerase gene. Pairwise sequence and phylogenetic analyses were performed using CLUSTAL V (DNASTAR) on the eight (A-H) standard full-length nucleotide sequences of HBV DNA from GenBank (NCBI) and the corresponding semi-nested PCR products from the HBV DNA polymerase gene. The differences in the semi-nested PCR fragments of the polymerase genes among genotypes A through F were greater than 4%, which is consistent with the intergenotypic divergence of at least 4% in HBV DNA S gene sequences. Genotyping using the semi-nested PCR products of the DNA polymerase genes revealed that only genotypes B, C, and D were present in the 50 cases, from Shenyang, China, with a distribution of 11 cases (22%), 25 cases (50%), and 14 cases (28%) respectively. These results demonstrate that our new genotyping method utilizing a fragment of the HBV DNA polymerase gene is valid and can be employed as a general genotyping strategy in areas with prevalent HBV genotypes A through F. In Shenyang, China, genotypes C, B, and D were identified with this new genotyping method, and genotype C was demonstrated to be the dominant genotype

The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 μM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 μM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development. © 2013 Tavis et al

Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.

Ledipasvir, a direct acting antiviral agent (DAA) targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H) that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants

³H-LDV binding to NS5A-6HIS.

<p><b>(A)</b> Each reaction, in a final volume of 200 μl, contained 50 nM of purified NS5A-6HIS and the indicated concentration of ³H-LDV in the absence () or presence (○) of unlabeled LDV. Bound ³H-LDV was measured as described in Materials and Methods. Each data point represents the average of 4–7 assays. <b>(B)</b> Specific binding of ³H-LDV to NS5A-6HIS (●) vs. NS5A-Y93H-6HIS (). Specific binding was defined as the difference between the amount of ³H-LDV bound in the absence (total binding) and presence (non-specific binding) of unlabeled LDV. Each data point represents the average of at least 3 assays.</p

Characterization of purified NS5A.

<p><b>(A)</b> Coomassie staining of purified proteins. Recombinant His-tagged proteins were purified as described in Materials and Methods. 1 μg of NS5A-6HIS and NS5A-Y93H-6HIS were subjected to SDS-PAGE and the protein bands were visualized by Coomassie Blue stain. Molecular weights for protein standards are indicated. <b>(B)</b> Phosphorylation sites determined by mass spectrometry of purified NS5A-6HIS and NS5A-Y93H-6HIS. Phosphorylation sites identified in NS5A-6HIS are shaded green; sites identified in NS5A-Y93H-6HIS are shaded red; and sites identified in both proteins are shaded cyan. <b>(C)</b> Analytical ultracentrifugation analysis of NS5A-6HIS, NS5A-Y93H-6HIS, and NS5A-domain 1. For NS5A-6HIS and NS5A-Y93H-6HIS, analytical ultracentrifugation was performed in 25 mM Tris pH 7.5, 150 mM NaCl, 0.01% NaN₃, 0.5 mM TCEP, 2 μg/ml Leupeptin, 0.02% C12E8, and 21.1% (v/v) D₂O. Sedimentation velocity analysis was performed at 42,000 rpm for two protein concentrations (3.3 and 6.6 μM). For NS5A-domain 1, analytical ultracentrifugation was performed in 25 mM Tris pH 8.0, 250 mM NaCl, 10% glycerol, and 0.5% DMSO. Sedimentation velocity analysis was performed at 48,000 rpm for 4 protein concentrations (7.5, 12, 15, 30 μM). Representative traces are shown for each protein. <b>(D)</b> Circular dichroism spectroscopy of NS5A-6HIS and NS5A-Y93H-6HIS. Circular dichroism measurements of NS5A-6HIS or NS5A-Y93H-6HIS (10 μM) were carried out at 20°C. The average of 3 measurements is shown.</p

BMS-Biotin does not compete for LDV binding to NS5A.

<p><b>(A)</b> Huh7-lunet cells were transfected with pTK-NS5A. 24 hr post transfection, cells were treated for 42 hr with a fixed concentration of BMS-Biotin and increasing concentrations of LDV or DCV. Cells were harvested and the membrane fraction was solubilized with C12E8. Solubilized proteins were incubated with streptavidin agarose beads and co-precipitated material was subjected to SDS-PAGE. NS5A was visualized by immunoblot analysis using an anti-NS5A antibody. <b>(B)</b> The relative intensity of pelleted NS5A, normalized to total NS5A, in (A) was quantified by densitometry. % Bound represents the amount of NS5A in the pellet relative to that in the control lane, which contained no competitor. <b>(C)</b>. Each reaction, in a final volume of 200 μl, contained 50 nM NS5A-6HIS, 30 nM ³H-LDV and the indicated concentration of unlabeled BMS-Biotin () or LDV (●). % Bound represents the amount of ³H-LDV bound relative to that in the control tube, which contained no unlabeled inhibitor. Each data point represents the average of at least 3 assays.</p

Competitive binding of ³H-LDV in the presence of unlabeled inhibitor.

<p>Each reaction, in a final volume of 200 μl, contained 50 nM NS5A-6HIS, 30 nM ³H-LDV and the indicated concentration of unlabeled LDV (●) or DCV (). % Bound represents the amount of ³H-LDV bound relative to that in the control tube, which contained no unlabeled inhibitor. Each data point represents the average of at least 3 assays. K_i was calculated using the Cheng-Prusoff equation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122844#pone.0122844.ref030" target="_blank">30</a>]. EC₅₀ represents the 50% effective inhibitory concentration of HCV RNA replication in the Renilla luciferase genotype 1b, Con 1 replicon cell line.</p