The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold.

Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) located in the 5' untranslated region of the genomic RNA that drives cap-independent initiation of translation of the viral message. The approximate secondary structure and minimum functional length of the HCV IRES are known, and extensive mutagenesis has established that nearly all secondary structural domains are critical for activity. However, the presence of an IRES RNA tertiary fold and its functional relevance have not been established. Using chemical and enzymatic probes of the HCV IRES RNA in solution, we show that the IRES adopts a unique three-dimensional structure at physiological salt concentrations in the absence of additional cofactors or the translation apparatus. Folding of the IRES involves cooperative uptake of magnesium and is driven primarily by charge neutralization. This tertiary structure contains at least two independently folded regions which closely correspond to putative binding sites for the 40 S ribosomal subunit and initiation factor 3 (eIF3). Point mutations that inhibit IRES folding also inhibit its function, suggesting that the IRES tertiary structure is essential for translation initiation activity. Chemical and enzymatic probing data and small-angle X-ray scattering (SAXS) experiments in solution show that upon folding, the IRES forms an extended structure in which functionally important loops are exposed. These results suggest that the 40 S ribosomal subunit and eIF3 bind an HCV IRES that is prefolded to spatially organize recognition domains.

Hepatitis C IRES: translating translation into a therapeutic target.

Several approaches have been undertaken in the attempt to inhibit hepatitis C virus (HCV) translation. Antisense oligonucleotides (AS ONs) have proven to be invaluable in the characterization of the HCV internal ribosome entry site (IRES). Chemical modification of oligonucleotides has resulted in optimized stability and specificity. Artificial ribozymes have also been developed to target the HCV IRES. Both techniques have demonstrated efficacy in vitro and in vivo. Various studies have identified cellular cofactor proteins that are required for IRES function, which may present themselves as intervention targets. Recent experiments have revealed that the HCV IRES uses a novel mechanism of recruiting translational components. These new advances in understanding the mechanism of HCV translation could lead to the development of novel IRES inhibitor strategies.

Mechanism of ribosome recruitment by hepatitis C IRES RNA.

Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 5' untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA-40S-eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.

Amantadine and rimantadine have no direct inhibitory effects against hepatitis C viral protease, helicase, ATPase, polymerase, and internal ribosomal entry site-mediated translation.

Amantadine, a drug known to inhibit influenza A viral matrix (M2) protein function, was reported to be an effective treatment in some patients with chronic hepatitis C virus (HCV) infection. Sequence comparison shows no homology between M2 and any of the HCV proteins. The effects of amantadine and a related analogue, rimantadine, on viral protease, helicase, ATPase, RNA-dependent RNA polymerase, and HCV internal ribosomal entry site (IRES) translation were tested by established in vitro biochemical assays. No inhibition (>15%) of HCV protease, helicase, ATPase, and polymerase was observed with concentrations up to 400 microgram/mL. IRES-specific inhibition was not observed at clinically relevant concentrations, but both cap and IRES reporter genes were suppressed at higher levels, suggesting nonspecific translation inhibition. In conclusion, amantadine and rimantadine have no direct and specific inhibitory effects against HCV protease, helicase, ATPase, polymerase, and IRES in vitro.

Hepatitis C virus internal ribosome entry site (IRES) stem loop IIId contains a phylogenetically conserved GGG triplet essential for translation and IRES folding.

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured RNA element that directs cap-independent translation of the viral polyprotein. Morpholino antisense oligonucleotides directed towards stem loop IIId drastically reduced HCV IRES activity. Mutagenesis studies of this region showed that the GGG triplet (nucleotides 266 through 268) of the hexanucleotide apical loop of stem loop IIId is essential for IRES activity both in vitro and in vivo. Sequence comparison showed that apical loop nucleotides (UUGGGU) were absolutely conserved across HCV genotypes and the GGG triplet was strongly conserved among related Flavivirus and Pestivirus nontranslated regions. Chimeric IRES elements with IIId derived from GB virus B (GBV-B) in the context of the HCV IRES possess translational activity. Mutations within the IIId stem loop that abolish IRES activity also affect the RNA structure in RNase T(1)-probing studies, demonstrating the importance of correct RNA folding to IRES function.

Enhancement of hepatitis C virus NS3 proteinase activity by association with NS4A-specific synthetic peptides: identification of sequence and critical residues of NS4A for the cofactor activity.

The NS3 proteinase of hepatitis C virus utilizes NS4A as a cofactor for cleavages at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region of the viral polyprotein. To characterize NS4A for its role in modulating the NS3 proteinase activity at various cleavage sites, synthetic peptides spanning various parts of NS4A were synthesized and tested in a cell-free trans-cleavage reaction using purified NS3 proteinase domain and polyprotein substrates. The NS3 proteinase domain was expressed in Escherichia coli, purified, denatured, and refolded to an enzymatically active form. We found that a 12-amino-acid peptide containing amino acid residues 22 to 33 in NS4A (CVVIVGRIVLSG) was sufficient for cofactor activity in NS3-mediated proteolysis. The peptide enhanced the cleavage at the NS5A/5B site and was necessary for NS3-mediated cleavage at NS4A/4B and NS4B/5A. Sequential amino acid substitution within the designated peptide identified residues I29 and I25 as critical for potential cofactor activity. We provide evidence that the NS4A peptide and the NS3 catalytic domain form an enzymatically active complex. These data suggest that the central 12-amino-acid peptide (aa 22-33) of NS4A is primarily important for the cofactor activity through complex formation with NS3, and the interaction may represent a new target for antiviral drug development.