Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.     

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

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.     

Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function

Translation of the hepatitis C virus (HCV) polyprotein is initiated at an internal ribosome entry site (IRES) element in the 5' untranslated region of HCV RNA. The HCV IRES element interacts directly with the 40S subunit, and biochemical experiments have implicated RNA elements near the AUG start codon as required for IRES-40S subunit complex formation. The data we present here show that two RNA stem loops, domains IIId and IIIe, are involved in IRES-40S subunit interaction. The structures of the two RNA domains were solved by NMR spectroscopy and reveal structural features that may explain their role in IRES function.     

Mutational and structural analysis of stem-loop IIIC of the hepatitis C virus and GB virus B internal ribosome entry sites

Translation of the open reading frames (ORF) of the hepatitis C virus (HCV) and closely related GB virus B (GBV-B) genomes is driven by internal ribosome entry site (IRES) elements located within the 5' non-translated RNA. The functioning of these IRES elements is highly dependent on primary and higher order RNA structures. We present here the solution structures of a common, critical domain within each of these IRESs, stem-loop IIIc. These ten-nucleotide hairpins have nearly identical sequences and similar overall tertiary folds. The final refined structure of each shows a stem with three G:C base-pairs and a novel tetraloop fold. Although the bases are buckled, the first and fourth nucleotides of both tetraloops form a Watson-Crick type base-pair, while the apical nucleotides are located in the major groove where they adopt C(2)-endo sugar puckering with B-form geometry. No hydrogen bonding interactions were observed involving the two apical residues of the tetraloop. Stability of the loops appears to be derived primarily from the stacking of bases, and the hydrogen bonding between the fourth and seventh residues. Mutational analysis shows that the primary sequence of stem-loop IIIc is important for IRES function and that the stem and first and fourth nucleotides of the tetraloop contribute to the efficiency of internal ribosome entry. Base-pair formation between these two positions is essential. In contrast, the apical loop nucleotides differ between HCV and GBV-B, and substitutions in this region of the hairpin are tolerated without major loss of function.     

Linker scanning mutagenesis of the internal ribosome entry site of poliovirus RNA.

The initiation of cap-independent translation of poliovirus mRNA occurs as a result of ribosome entry at an internal site(s) within the 5' noncoding region. A series of linker scanning mutations was constructed to define the genetic determinants of RNA-protein interactions that lead to high-fidelity translation of this unusual viral mRNA. The mutations are located within two distinct stem-loop structures in the 5' noncoding region of poliovirus RNA that constitute a major portion of a putative internal ribosome entry site. On the basis of our data derived from genetic and biochemical assays, the stability of one of the stem-loop structures appears to be essential for translation initiation via internal binding of ribosomes. However, the second stem-loop structure may function in a manner that requires base pairing and proper spacing between specific nucleotide sequences. By employing RNA electrophoretic mobility shift assays, an RNA-protein interaction was detected for this latter stem-loop structure that does not occur in RNAs containing mutations which perturb the predicted hairpin structure. Analysis of in vivo-selected virus revertants, in combination with mobility shift assays, suggests that extensive genetic rearrangement can lead to restoration of 5' noncoding region functions, possibly by the repositioning of specific RNA sequence or structure motifs.     

2-Aminobenzoxazole ligands of the hepatitis C virus internal ribosome entry site

2-Aminobenzoxazoles have been synthesized as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The compounds were designed to explore the less basic benzoxazole system as a replacement for the core scaffold in previously discovered benzimidazole viral translation inhibitors. Structure–activity relationships in the target binding of substituted benzoxazole ligands were investigated.     

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA

Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5' untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA(f)-binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.     

Structural characterization of three RNA hexanucleotide loops from the internal ribosome entry site of polioviruses.

Structural characteristics of three RNA hairpins from the internal ribosome entry site of poliovirus mRNAs have been determined in solution by NMR. Complete proton, phosphorus and carbon resonance assignments were made for the three 16 nt hairpins. The loop sequences, 5'-AAUCCA , AAACCA and GAACCA, have been shown to be essential for viral mRNA translation. NOESY spectra for the three oligomers were very similar indicating a common three dimensional structure. Stems were A-type duplexes with C3'-endo sugar pucker. In the loops, sequential base stacking interactions were detected for all bases except between U8/A8 and C9, indicating a turn in the phosphodiester backbone at this point. Only one nucleotide, U8/A8, had a sugar pucker which deviated appreciably from C3'-endo. The final base in the loop, A11, exhibited an unusual gauche (-) gamma angle. An ensemble of 10 structures calculated for one hairpin using restrained molecular dynamics shows that the first three bases of the loop are turned so as to be exposed to the exterior of the molecule, while the remaining three bases are in an orientation approximating a continuation of the stem helix. Structure calculations and NMR relaxation measurements indicate that the loop apex is subject to considerable local dynamics.     

The hepatitis C virus RNA 3'-untranslated region strongly enhances translation directed by the internal ribosome entry site

The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5'- and 3'-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5'-UTR, and subsequent viral RNA replication requires sequences in the 3'-UTR and in the 5'-UTR. Addressing previous conflicting reports on a possible function of the 3'-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3'-UTR function. A translation enhancer function of the HCV 3'-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3'-UTR with a precise 3' terminus. The 3'-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U . C) tract, and the most 3' terminal stem-loop 1 of the highly conserved 3' X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3' X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3'-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3'-5' end interaction and a switch between translation and RNA replication.     

RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

Here, we show that Escherichia coli Ribonuclease III cleaves specifically the RNA genome of hepatitis C virus (HCV) within the first 570 nt with similar efficiency within two sequences which are ~400 bases apart in the linear HCV map. Demonstrations include determination of the specificity of the cleavage sites at positions C²⁷ and U³³ in the first (5′) motif and G⁴³⁹ in the second (3′) motif, complete competition inhibition of 5′ and 3′ HCV RNA cleavages by added double-stranded RNA in a 1:6 to 1:8 weight ratio, respectively, 50% reverse competition inhibition of the RNase III T7 R1.1 mRNA substrate cleavage by HCV RNA at 1:1 molar ratio, and determination of the 5′ phosphate and 3′ hydroxyl end groups of the newly generated termini after cleavage. By comparing the activity and specificity of the commercial RNase III enzyme, used in this study, with the natural E.coli RNase III enzyme, on the natural bacteriophage T7 R1.1 mRNA substrate, we demonstrated that the HCV cuts fall into the category of specific, secondary RNase III cleavages. This reaction identifies regions of unusual RNA structure, and we further showed that blocking or deletion of one of the two RNase III-sensitive sequence motifs impeded cleavage at the other, providing direct evidence that both sequence motifs, besides being far apart in the linear RNA sequence, occur in a single RNA structural motif, which encloses the HCV internal ribosome entry site in a large RNA loop.     

A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III-IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId

The hepatitis C virus (HCV) has a positive single-stranded RNA genome, and translation starts within the internal ribosome entry site (IRES) in a cap-independent manner. The IRES is well conserved among HCV subtypes and has a unique structure consisting of four domains. We used an in vitro selection procedure to isolate RNA aptamers capable of binding to the IRES domains III–IV. The aptamers that were obtained shared the consensus sequence ACCCA, which is complementary to the apical loop of domain IIId that is known to be a critical region of IRES-dependent translation. This convergence suggests that domain IIId is preferentially selected in an RNA–RNA interaction. Mutation analysis showed that the aptamer binding was sequence and structure dependent. One of the aptamers inhibited translation both in vitro and in vivo. Our results indicate that domain IIId is a suitable target site for HCV blockage and that rationally designed RNA aptamers have great potential as anti-HCV drugs.     

A small stem loop element directs internal initiation of the URE2 internal ribosome entry site in Saccharomyces cerevisiae

Internal initiation of translation is the process of beginning protein synthesis independent of the m(7)G cap structure at the 5'-end of an mRNA molecule. We have previously shown that the URE2 mRNA in the yeast Saccharomyces cerevisiae contains an internal ribosome entry site (IRES) whose activity is suppressed by eukaryotic initiation factor 2A (eIF2A; YGR054W). In this study, the minimal sequence required to efficiently direct internal initiation was determined using a system that abrogates cap-dependent scanning of the 40 S ribosomal subunit in both wild-type and eIF2A knock-out cells. Subsequently, secondary structural elements within the minimal sequence were determined by probing with RNases T1 and V1 and the small molecule diethylpyrocarbonate. It was found that the URE2 minimal IRES comprises a 104 nucleotide A-rich stem loop element encompassing the internal AUG codon. Interestingly, the internal AUG seems to be involved in base-pairing interactions that would theoretically hamper its ability to interact with incoming initiator tRNA molecules. Furthermore, none of the truncations used to identify the minimal IRES element were capable of abrogating the suppressive effect of eIF2A. Our data provide the first insight into the RNA structural requirements of the yeast translational machinery for cap-independent initiation of protein synthesis.     

A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo.

Mutants of foot-and-mouth disease virus (FMDV) with altered biological properties can be selected during the course of persistent infection of BHK-21 cells with FMDV C-S8c1 (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). Two nucleotide substitutions, U to C at position -376 and A to G at position -15, (counting as +1 the A of the first functional AUG), were fixed within the internal ribosome entry site (IRES) of R100, the virus rescued after 100 passages of the carrier BHK-21 cells. IRES-directed cap-independent protein synthesis was quantitated by using bicistronic constructs of the form chloramphenicol acetyltransferase gene-IRES-luciferase gene. The IRES from R100 was 1.5- to 5-fold more active than that of C-S8c1 in directing cap-independent luciferase synthesis. This enhanced translational activity was observed when the RNAs were transcribed either in the nucleus or in the cytoplasm by a weak or a strong promoter, respectively. C-S8c1 and R100 IRES elements were functional in both FMDV-sensitive and FMDV-resistant cells (including persistently infected R cells), indicating that factors mediating cap-independent protein synthesis are not limited in any of the analyzed cell lines. Constructs in which each of the two mutations in the R100 IRES were analyzed separately indicate that the transition at position -376 is responsible for the enhanced activity of the R100 IRES. By estimating the effect that an increase in the initial translation efficiency may have on subsequent RNA replication steps, we suggest that the modifications in the IRES elements can account for the previously described hypervirulence of FMDV R100 for BHK-21 cells. The results show that a single point mutation in an IRES element of a picornavirus can cause an increase in translation efficiency.     

Interaction of translation initiation factor eIF4B with the poliovirus internal ribosome entry site

Poliovirus translation is initiated at the internal ribosome entry site (IRES). Most likely involving the action of standard initiation factors, this highly structured cis element in the 5" noncoding region of the viral RNA guides the ribosome to an internal silent AUG. The actual start codon for viral protein synthesis further downstream is then reached by ribosomal scanning. In this study we show that two of the secondary structure elements of the poliovirus IRES, domain V and, to a minor extent, domain VI, are the determinants for binding of the eukaryotic initiation factor eIF4B. Several mutations in domain V which are known to greatly affect poliovirus growth also seriously impair the binding of eIF4B. The interaction of eIF4B with the IRES is not dependent on the presence of the polypyrimidine tract-binding protein, which also binds to the poliovirus IRES. In contrast to its weak interaction with cellular mRNAs, eIF4B remains tightly associated with the poliovirus IRES during the formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. These results indicate that the interaction of eIF4B with the 3" region of the poliovirus IRES may be directly involved in translation initiation.     

A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy

The 5′ untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II–IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI. The 574 nt-long molecule analyzed underwent an unexpected, Mg²⁺-induced switch between two alternative conformations: from ‘open’, elongated morphologies at 0–2 mM Mg²⁺ concentration to a ‘closed’, comma-shaped conformation at 4–6 mM Mg²⁺. This sharp transition, confirmed by gel-shift analysis and partial RNase T1 cleavage, was hindered by the microRNA miR-122. The comma-shaped IRES-574 molecules visualized at 4–6 mM Mg²⁺ in the absence of miR-122 showed two arms. Our data support that the first arm would contain domain III, while the second one would be composed of domains (I–II)+(V–VI) thanks to a long-range RNA interaction between the I-II spacer and the basal region of domain VI. This reinforces the previously described structural continuity between the HCV IRES and its flanking domains I, V and VI.     

Mutation of a single conserved nucleotide between the cloverleaf and internal ribosome entry site attenuates poliovirus neurovirulence

The chemical synthesis of poliovirus (PV) cDNA combined with the cell-free synthesis of infectious particles yielded virus whose mouse neurovirulence was highly attenuated (J. Cello, A. V. Paul, and E. Wimmer, Science 297:1016-1018, 2002). Compared to the wild-type PV1 (Mahoney) [PV1(M)] sequence, the synthetic virus genome harbored 27 nucleotide (nt) changes deliberately introduced as genetic markers. Of the 27 nucleotide substitutions, the UA-to-GG exchanges at nucleotides 102/103, mapping to a region between the cloverleaf and the internal ribosome entry site (IRES) in the 5'-nontranslated region, were found to be involved in the observed attenuation phenotype in mice. The UA/GG mutation at nt 102/103 in the synthetic PV1(M) [sPV1(M)] background conferred also a ts phenotype of replication to the virus in human neuroblastoma cells. Conversely, the exchange of GG to wild-type (wt) UA at 102/103 in an sPV1(M) background restored wt neurovirulence in CD155 transgenic (tg) mice and suppressed the ts phenotype in SK-N-MC cells. All poliovirus variants replicated well in HeLa cells at the two temperatures, regardless of the sequence at the 102/103 locus. Analyses of variants isolated from sPV(M)-infected CD155 tg mice revealed that the G(102)G(103)-to-G(102)A(103) reversion alone reestablished the neurovirulent phenotype. This suggests that a single mutation is responsible for the observed change of the neurovirulence phenotype. sPV1(M) RNA is translated in cell extracts of SK-N-MC cells with significantly lower efficiency than PV1(M) RNA or sPV1(M) RNA with a G(102)-to-A(102) reversion. These studies suggest a function for the conserved nucleotide (A(103)) located between the cloverleaf and the IRES which is important for replication of PV in the central nervous system of CD155 tg mice and in human cells of neuronal origin.     

La autoantigen is required for the internal ribosome entry site-mediated translation of Coxsackievirus B3 RNA

Translation initiation in Coxsackievirus B3 (CVB3) occurs via ribosome binding to an internal ribosome entry site (IRES) located in the 5′-untranslated region (UTR) of the viral RNA. This unique mechanism of translation initiation requires various trans-acting factors from the host. We show that human La autoantigen (La) binds to the CVB3 5′-UTR and also demonstrate the dose-dependent effect of exogenously added La protein in stimulating CVB3 IRES-mediated translation. The requirement of La for CVB3 IRES mediated translation has been further demonstrated by inhibition of translation as a result of sequestering La and its restoration by exogenous addition of recombinant La protein. The abundance of La protein in various mouse tissue extracts has been probed using anti-La antibody. Pancreatic tissue, a target organ for CVB3 infection, was found to have a large abundance of La protein which was demonstrated to interact with the CVB3 5′-UTR. Furthermore, exogenous addition of pancreas extract to in vitro translation reactions resulted in a dose dependent stimulation of CVB3 IRES-mediated translation. These observations indicate the role of La in CVB3 IRES-mediated translation, and suggest its possible involvement in the efficient translation of the viral RNA in the pancreas.