A potential RNA drug target in the hepatitis C virus internal ribosomal entry site

Subdomain IIId from the hepatitis C virus (HCV) internal ribosome entry sit e (IRES) has been shown to be essential for cap-independent translation. We have conducted a structural study of a 27-nt fragment, identical in sequen ce to IIId, to explore the structural features of this subdomain, The propo sed secondary structure of IIId is comprised of two 3 bp helical regions se parated by an internal loop and closed at one end by a 6-nt terminal loop. NMR and molecular modeling were used interactively to formulate a validated model of the three-dimensional structure of IIId. We found that this fragm ent contains several noncanonical structural motifs and non-Watson-Crick ba se pairs, some of which are common to other RNAs, In particular, a motif ch aracteristic of the rRNA alpha -sarcin/ricin loop was located in the intern al loop. The terminal loop, 5'-UUGGGU, was found to fold to form a trinucle otide loop closed by a trans-wobble U(.)G base pair. The sixth nucleotide w as bulged out to allow stacking of this U(.)G pair on the adjacent helical region. In vivo mutational analysis in the context of the full IRES confirm ed the importance of each structural motif within IIId for IRES function. T hese findings may provide clues as to host cellular proteins that play a ro le in IRES-directed translation and, in particular, the mechanism through w hich host ribosomes are sequestered for viral function.