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Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA.

ABSTRACT: The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.

SUBMITTER: Zhou S

PROVIDER: S-EPMC3758467 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA.

Zhou Shou S Rynearson Kevin D KD Ding Kejia K Brunn Nicholas D ND Hermann Thomas T

Bioorganic & medicinal chemistry 20130402 20

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay fo ...[more]

PMID: 23602522

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Project description:Multiple processes exist in a cell to ensure continuous production of essential proteins either through cap-dependent or cap-independent translation processes. Viruses depend on the host translation machinery for viral protein synthesis. Therefore, viruses have evolved clever strategies to use the host translation machinery. Earlier studies have shown that genotype 1 hepatitis E virus (g1-HEV) uses both cap-dependent and cap-independent translation machineries for its translation and proliferation. Cap-independent translation in g1-HEV is driven by an 87-nucleotide-long RNA element that acts as a noncanonical, internal ribosome entry site-like (IRESl) element. Here, we have identified the RNA-protein interactome of the HEV IRESl element and characterized the functional significance of some of its components. Our study identifies the association of HEV IRESl with several host ribosomal proteins, demonstrates indispensable roles of ribosomal protein RPL5 and DHX9 (RNA helicase A) in mediating HEV IRESl activity, and establishes the latter as a bona fide internal translation initiation site. IMPORTANCE Protein synthesis is a fundamental process for survival and proliferation of all living organisms. The majority of cellular proteins are produced through cap-dependent translation. Cells also use a variety of cap-independent translation processes to synthesize essential proteins during stress. Viruses depend on the host cell translation machinery to synthesize their own proteins. Hepatitis E virus (HEV) is a major cause of hepatitis worldwide and has a capped positive-strand RNA genome. Viral nonstructural and structural proteins are synthesized through a cap-dependent translation process. An earlier study from our laboratory reported the presence of a fourth open reading frame (ORF) in genotype 1 HEV, which produces the ORF4 protein using a cap-independent internal ribosome entry site-like (IRESl) element. In the current study, we identified the host proteins that associate with the HEV-IRESl RNA and generated the RNA-protein interactome. Through a variety of experimental approaches, our data prove that HEV-IRESl is a bona fide internal translation initiation site.

Dataset Information

Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA.

Publications

Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA.

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