Inhibition of the ATPase activity of the chaperone protein HSP90 is a potential strategy for treatment of cancers. We have determined structures of the HSP90alpha N-terminal domain complexed with the purine-based inhibitor, PU3, and analogs with enhanced potency both in enzyme and cell-based assays. The compounds induce upregulation of HSP70 and downregulation of the known HSP90 client proteins Raf-1, CDK4, and ErbB2, confirming that the molecules inhibit cell growth by a mechanism dependent on HSP90 inhibition. We have also determined the first structure of the N-terminal domain of HSP90beta, complexed with PU3. The structures allow a detailed rationale to be developed for the observed affinity of the PU3 class of compounds for HSP90 and also provide a structural framework for design of compounds with improved binding affinity and drug-like properties.

Subdomain IIId from the hepatitis C virus (HCV) internal ribosome entry site (IRES) has been shown to be essential for cap-independent translation. We have conducted a structural study of a 27-nt fragment, identical in sequence to IIId, to explore the structural features of this subdomain. The proposed secondary structure of IIId is comprised of two 3 bp helical regions separated 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 fragment contains several noncanonical structural motifs and non-Watson-Crick base pairs, some of which are common to other RNAs. In particular, a motif characteristic of the rRNA a-sarcin/ricin loop was located in the internal loop. The terminal loop, 59-UUGGGU, was found to fold to form a trinucleotide loop closed by a trans-wobble U‫ؠ‬ ‫ؠ‬ ‫ؠ‬G base pair. The sixth nucleotide was bulged out to allow stacking of this U‫ؠ‬ ‫ؠ‬ ‫ؠ‬G pair on the adjacent helical region. In vivo mutational analysis in the context of the full IRES confirmed the importance of each structural motif within IIId for IRES function. These findings may provide clues as to host cellular proteins that play a role in IRES-directed translation and, in particular, the mechanism through which host ribosomes are sequestered for viral function.

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is recognized specifically by the small ribosomal subunit and eukaryotic initiation factor 3 (eIF3) before viral translation initiation. Using extensive mutagenesis and structure probing analysis, we show that the eIF3-binding domain of the HCV IRES contains an internal loop structure (loop IIIb) and an adjacent mismatched helix that are important for IRES-dependent initiation of translation. NMR studies reveal a unique three-dimensional structure for this internal loop that is conserved between viral isolates of varying primary sequence in this region. These data indicate that internal loop IIIb may be an attractive target for structure-based design of new antiviral agents.

The 5h untranslated region (5hUTR) of hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) which directs translation of the viral open reading frame (ORF). The 5hUTR is highly conserved between virus isolates in both primary sequence and predicted secondary structure. We cloned and sequenced the 5h regions (nt 18 of the 5hUTR to nt 15 of the core coding sequence) of HCV isolates representing the six major genotypes and subcloned these into a bicistronic, dual luciferase reporter construct. The relative expression of the two luciferases, one directed by the HCV IRES and the other by cap-dependent ribosome scanning,

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