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Inhibitor-induced structural change in the HCV IRES domain IIa RNA.

ABSTRACT: Translation of the hepatitis C virus (HCV) RNA is initiated from a highly structured internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) of the RNA genome. An important structural feature of the native RNA is an approximately 90 degrees helical bend localized to domain IIa that positions the apical loop of domain IIb of the IRES near the 40S ribosomal E-site to promote eIF2-GDP release, facilitating 80S ribosome assembly. We report here the NMR structure of a domain IIa construct in complex with a potent small-molecule inhibitor of HCV replication. Molecular dynamics refinement in explicit solvent and subsequent energetic analysis indicated that each inhibitor stereoisomer bound with comparable affinity and in an equivalent binding mode. The in silico analysis was substantiated by fluorescence-based assays showing that the relative binding free energies differed by only 0.7 kcal/mol. Binding of the inhibitor displaces key nucleotide residues within the bulge region, effecting a major conformational change that eliminates the bent RNA helical trajectory, providing a mechanism for the antiviral activity of this inhibitor class.

SUBMITTER: Paulsen RB

PROVIDER: S-EPMC2867761 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Inhibitor-induced structural change in the HCV IRES domain IIa RNA.

Paulsen Ryan B RB Seth Punit P PP Swayze Eric E EE Griffey Richard H RH Skalicky Jack J JJ Cheatham Thomas E TE Davis Darrell R DR

Proceedings of the National Academy of Sciences of the United States of America 20100401 16

Translation of the hepatitis C virus (HCV) RNA is initiated from a highly structured internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) of the RNA genome. An important structural feature of the native RNA is an approximately 90 degrees helical bend localized to domain IIa that positions the apical loop of domain IIb of the IRES near the 40S ribosomal E-site to promote eIF2-GDP release, facilitating 80S ribosome assembly. We report here the NMR structure of a domain IIa c ...[more]

PMID: 20360559

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Project description:The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-β, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism.

Dataset Information

Inhibitor-induced structural change in the HCV IRES domain IIa RNA.

Publications

Inhibitor-induced structural change in the HCV IRES domain IIa RNA.

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