Hepatitis C virus internal ribosome entry site RNA contains a tertiary structural element in a functional domain of stem-loop II

Abstract

The internal ribosome entry site (IRES) of hepatitis C virus (HCV) RNA contains >300 bases of highly conserved 5'-terminal sequence, most of it in the uncapped 5'-untranslated region (5'-UTR) upstream from the single AUG initiator triplet at which translation of the HCV polyprotein begins. Although progress has been made in defining singularities like the RNA pseudoknot near this AUG, the sequence and structural features of the HCV IRES which stimulate accurate and efficient initiation of protein synthesis are only partially defined. Here we report that a region further upstream from the AUG, stem-loop II of the HCV IRES, also contains an element of local tertiary structure which we have detected using RNase H cleavage and have mapped using the singular ability of two bases therein to undergo covalent intra-chain crosslinking stimulated by UV light. This pre-existing element maps to two non-contiguous stretches of the HCV IRES sequence, residues 53-68 and 103-117. Several earlier studies have shown that the correct sequence between bases 45 and 70 of the HCV IRES stem-loop II domain is required for initiation of protein synthesis. Because features of local tertiary structure like the one we report here are often associated with protein binding, we propose that the HCV stem-loop II element is directly involved in IRES action.

Structural analysis of hepatitis C RNA genome using DNA microarrays

“…Images shown in Figure 2 have been reproducibly observed in two independent experiments obtained by means of different labelled RNA transcript preparations. These results are in agreement with previous ones using RNase H and DNA (43)(44)(45), and demonstrate the suitability of the proposed method to distinguish highly structured RNA from less structured ones, in the HCV genome. Macroarrays were printed manually and compared by visual inspection and therefore not used for fine quantitation purposes but they suggested that the approach was valid and we could go to the next step using microarrays.…”

“…These oligonucleotides were complementary to specific viral regions in which the genomic RNA adopts different degrees of structure, as previously measured by means of RNase H accessibility studies (44,45) (Figure 1A). …”

“…Hybridization of the labelled transcripts was carried out under non-denaturing conditions, using appropriate temperature and buffer composition to allow binding of the RNA transcript to the immobilized probes, without disturbing its secondary/tertiary structure (44). Hybridization mix consisted of 60 nM labelled RNA transcript, 1· buffer H (20 mM Hepes-HCl pH 7.8, 50 mM KCl, 10 mM MgCl 2 , 1 mM DTT), 1· BSA, 50 mg/ml tRNA, 50 mg/ml poly A, 0.01% SDS and 40 U RNasin.…”

“…In previous studies, structural features of HCV RNA of two different genomic regions, 5 0 NCR (43,44) and NS2 (45), have been analysed using complementary deoxyoligonucleotide probes and RNase H cleavage. Though this method has proven to be a powerful tool to map RNA structural degree and to determine accessible sites for ribozyme targeting, it is very laborious and time consuming.…”

Searching for IRES

“…The initial secondary structure had been predicted using both enzymatic probing results as constraints in an early version of the MFOLD program (Zuker and Stiegler 1981) along with comparative computer-predicted models with the UTRs of BVDV and CSFV (Brown et al 1992). There have been numerous studies adding to the refinement of the IRES structure (Lemon and Honda 1997;Lyons et al 2001;Odreman-Macchioli et al 2001), arriving at two similar models (Honda et al 1999;Zhao and Wimmer 2001;Lytle et al 2002) with some differences in domain II. Smaller stem-loop motifs of the HCV IRES structure have had their tertiary structure determined using both X-ray crystallography (Kieft et al 2002) and NMR (Klinck et al 2000;Lukavsky et al 2000;Collier et al 2002).…”