Development of intergenotypic chimeric replicons to determine the broad-spectrum antiviral activities of hepatitis C virus polymerase inhibitors - PubMed (original) (raw)
Development of intergenotypic chimeric replicons to determine the broad-spectrum antiviral activities of hepatitis C virus polymerase inhibitors
Koleen J Herlihy et al. Antimicrob Agents Chemother. 2008 Oct.
Abstract
To address the need for broad-spectrum antiviral activity characterization of hepatitis C virus (HCV) polymerase inhibitors, we created a panel of intergenotypic chimeric replicons containing nonstructural (NS) protein NS5B sequences from genotype 2b (GT2b), GT3a, GT4a, GT5a, and GT6a HCV isolates. Viral RNA extracted from non-GT1 HCV patient plasma was subjected to reverse transcription. The NS5B region was amplified by nested PCR and introduced into the corresponding region of the GT1b (Con-1) subgenomic reporter replicon by Splicing by Overlap Extension (SOEing) PCR. Stable cell lines were generated with replication-competent chimeras for in vitro antiviral activity determination of HCV nonnucleoside polymerase inhibitors (NNIs) that target different regions of the protein. Compounds that bind to the NNI2 (thiophene carboxylic acid) or NNI3 (benzothiadiazine) allosteric sites showed 8- to >1,280-fold reductions in antiviral activity against non-GT1 NS5B chimeric replicons compared to that against the GT1b subgenomic replicon. Smaller reductions in susceptibility, ranging from 0.2- to 33-fold, were observed for the inhibitor binding to the NNI1 (benzimidazole) site. The inhibitor binding to the NNI4 (benzofuran) site showed broad-spectrum antiviral activity against all chimeric replicons evaluated in this study. In conclusion, evaluation of HCV NNIs against intergenotypic chimeric replicons showed differences in activity spectrum for inhibitors that target different regions of the enzyme, some of which could be associated with specific residues that differ between GT1 and non-GT1 polymerases. Our study demonstrates the utility of chimeric replicons for broad-spectrum activity determination of HCV inhibitors.
Figures
FIG. 1.
Structures of HCV protease and polymerase inhibitors used in this study.
FIG. 2.
Alignment of the HCV polymerase sequences utilized in this study. Amino acid residues 419 and 482, located in the thumb domain of the polymerase (25), are highlighted in gray boxes. The key amino acid residues that are present in the active and NNI-binding sites are also highlighted.
FIG. 3.
Replication fitness of intergenotypic chimeric replicons in transient replication (A) and colony formation (B) assays. (A) HCV reporter replicon constructs were electroporated into Huh7.5 cells, seeded in 96-well plates at 6 × 104 cells/well, and cultured for 3 days before RLuc activity was measured. Results represent the averages of three to five individual experiments, and error bars represent standard deviations. (B) 8 × 106 Huh7.5 cells were electroporated with 1 μg reporter replicon RNA and seeded in 10-cm dishes. Colonies were selected for 3 weeks in 250 μg/ml of G418 and stained with crystal violet.
FIG. 4.
The NNI-binding sites of the HCV polymerase. The X-ray crystal structure of the 1b-BK NS5BΔ21 protein is shown (27, 36), with protein backbone in a ribbon structure and the key amino acid residues that are present in the active and NNI-binding sites indicated and highlighted in yellow. Amino acid residues L419 and I482, located in the thumb domain of the polymerase, are also indicated.
References
- Ago, H., T. Adachi, A. Yoshida, M. Yamamoto, N. Habuka, K. Yatsunami, and M. Miyano. 1999. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure 7:1417-1426. - PubMed
- Carroll, S. S., and D. B. Olsen. 2006. Nucleoside analog inhibitors of hepatitis C virus replication. Infect. Disord. Drug Targets 6:17-29. - PubMed
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