HCV is a positive-sense single-strand RNA virus, classified in the genus hepacivirus, family Flaviviridae and possesses an internal ribosomal entry site (IRES) at the 5' end of its genome. This IRES element, which closely resembles those of pestiviruses, is a complex RNA structure containing distinct domains and can interact specifically with the ribosomal subunits, positioning them directly over the initiation codon. Furthermore, the RNA structure around the initiation codon affects the HCV IRES efficiency significantly. The RNA structure-dependence of the HCV IRES-mediated initiation is unique among all eukaryotic mRNAs, and is reminiscent of prokaryotic mechanisms. HCV IRES-mediated initiation is an integral part of the viral replication mechanism and, due to its specificity and sensitivity to minor structural changes, is considered one of the best targets for antiviral strategies. It also provides an exceptional opportunity for the study of RNA structure/function and basic translation initiation mechanism in eukaryotes.
A number of RNA-binding compounds were screened for their effect on the HCV IRES-mediated initiation in in vitro translation experiments. Cobalamin compounds inhibited this mechanism without affecting initiation of cap-dependent or two other viral IRES-dependent mRNAs. Notably, cyanocobalamin could distinguish between the HCV and extremely similar classical swine fever virus- (a pestivirus) IRESs. These findings plus the effect of magnesium ion concentration on the observed inhibition suggested a highly structure-specific interaction between cyanocobalamin and HCV IRES.
A selectable eukaryotic bicistronic vector was constructed and used to clone stable indicator cell lines of hepatic origin. These indicator cells can be used for the screening of potential inhibitory compounds on the HCV IRES-mediated initiation. However, the study of cyanocobalamin is still hindered by the impermeability of the cell membranes to cyanocobalamin.
Titration experiments suggested that a single binding event was responsible for the cyanocobalamin-induced inhibition. The calculated dissociation constant was high, causing difficulties in the detection of a specific HCV IRES-cyanocobalamin interaction. Furthermore, HCV IRES is a highly complex RNA structure that may act as a polymeric acceptor binding multiple cyanocobalamin molecules. Thus, it was necessary to map the site of this putative interaction. Since structural modifications of IRES alter its translational efficiency, a novel competition experiment was designed. Nonspecific translational inhibition of reporter constructs by the addition of a competitor IRES element plus cyanocobalamin suggested that ribosomal complexes are "trapped" by cyanocobalamin on the HCV IRES. This trapping effect was dose-dependent and specific to the HCV IRES. Further studies indicated that the presence of domain IV is required for the trapping effect.
A ribosomal toeprinting experiment was developed to study the initiation complex formation on the HCV IRES in the presence of various translational inhibitors. Consistent with the "trapping" theory, cyanocobalamin enhanced the ribosomal toeprint specifically on the HCV IRES. This finding was confirmed by a toeprinting-based competition experiment. Finally, a gradient centrifugation analysis showed that cyanocobalamin indeed causes the trapping of ribosomal complexes on the HCV IRES in vitro, and the complexes trapped are in the 8OS stage.
The effect of cyanocobalamin on the HCV IRES structure was demonstrated by an RNase probing experiment. The results suggested that a tertiary interaction in domain IV (a new pseudoknot) was stabilized by cyanocobalamin, resulting in a lowered accessibility of the first and second codon of the HCV ORF to the ribosome. A mutational analysis showed that stabilization of the proposed tertiary interaction resulted in an inhibition of translation, while destabilization caused a dramatic increase in translation efficiency. These results were consistent with previous findings and were utilized to propose a model for a novel translational control mechanism in HCV.
Cobalamins, which are abundant in hepatocytes, may play a regulatory role in the replication of HCV. The structure-specific interaction of cyanocobalamin with the HCV IRES provides an exceptional opportunity to study the effects of a small ligand on the structure of a highly specialized complex RNA element. Moreover the effect of this interaction on the initiation process can be used as a template for the development of new anti-HCV therapeutics. These studies can also shed light on the regulatory mechanisms that determine the fate of this virus during one of its most susceptible replication stages.