Structure of a hepatitis C virus RNA domain in complex with a translation inhibitor reveals a binding mode reminiscent of riboswitches

Abstract

The internal ribosome entry site (IRES) in the hepatitis C virus (HCV) RNA genome is essential for the initiation of viral protein synthesis. IRES domains adopt well-defined folds that are potential targets for antiviral translation inhibitors. We have determined the three-dimensional structure of the IRES subdomain IIa in complex with a benzimidazole translation inhibitor at 2.2 Å resolution.Comparison to the structure of the unbound RNA in conjunction with studies of inhibitor binding to the target in solution demonstrate that the RNA undergoes a dramatic ligand-induced conformational adaptation to form a deep pocket that resembles the substrate binding sites in riboswitches. The presence of a well-defined ligand-binding pocket within the highly conserved IRES subdomain IIa holds promise for the development of unique anti-HCV drugs with a high barrier to resistance.crystallography | hepatitis C virus inhibitor | RNA structure I nfection with hepatitis C virus (HCV), which affects over 170 million individuals worldwide, is a leading cause of liver failure and hepatocellular carcinoma (1). Until earlier this year, when two protease inhibitors were approved as the first direct antiviral drugs for the treatment of HCV infection (2), the standard anti-HCV therapy consisted of an immunostimulatory regimen of pegylated interferon-α and the nucleoside analog ribavirin, which suffered from low efficacy as well as serious side effects (3). The prevalence of preexisting drug-resistance mutations in HCV quasispecies due to the low fidelity of the viral RNA-dependent RNA polymerase (NS5B) creates an urgent need for combination therapy with unique antiviral agents directed at distinct HCV targets (4).Among the potential targets for HCV inhibitors, the 5′ untranslated region (UTR) of the viral RNA genome stands out for its high sequence conservation within virus clinical isolates (5), which exceeds the conservation of the HCV protein reading frames. The HCV 5′ UTR harbors an internal ribosome entry site (IRES) which recruits host cell 40S ribosomal subunits and ultimately initiates translation of virus proteins via a 5′ cap-independent mechanism (6, 7). The function of the IRES relies on a structured RNA element, which contains several independently folding domains (Fig. 1A) (8, 9). The three-dimensional structure of the subdomain IIa target was previously determined in our laboratory revealing an overall bent architecture around an RNA internal loop (Fig. 1B) (10), in agreement with NMR analyses of the full domain II (11) and cryoelectron microscopy studies of 13). The L-shaped conformation of subdomain IIa directs the apical hairpin loop IIb toward the ribosomal E site in proximity of the active site. Ribosomal association of domain II induces a conformational change in the 40S head and closes the mRNA binding cleft. Both, the correct positioning of the viral mRNA initiation codon as well as the joining of the ribosomal subunits to form functional 80S units depend critically on the L-shaped architecture of the domain II (7...

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

“…Isis-11 showed binding to a chimeric HCV subdomain IIa bulge, leading to a conformational change that widens the interhelical angle. 30,35,45 It remains to be established whether there is a different flexibility of the bulges of HCV IIa and FMDV 3a subdomains that may account for the observed differences in binding affinities.…”

“…This result suggests that the 2 inhibitor compounds likely induce similar conformational changes in the HCV IRES subdomain IIa. 30,35 To assess the target selectivity of IRAB and Isis-11 for both HCV and FMDV IRES subdomains, fluorescence titrations were also performed in the presence of a 10-fold molar excess of competitor tRNA mix , which corresponds to about 27-fold nucleotide excess in the case of SL3A/AP167 and 23-fold in the case of SLIIa/AP54. The EC 50 determined for the mixtures of tRNA and either SL3A/AP167 or SLIIa/AP54 are higher than those found for the oligonucleotides alone, as often in this kind of competition experiments.…”

“…6A), used earlier for binding assays of Isis-11, 30 was also employed to assess IRAB affinity. Not Red letters depict nts with absolute SHAPE differences higher than 0.3; font size reflects the magnitude of SHAPE difference.…”

“…27 Recently, a 2-aminobenzimidazole library has been screened for binding to RNA internal loops, 28 and a bisbenzimidazole derivative has been reported to bind the expanded r(CUG) repeats that cause myotonic dystrophy type 1 (DM1), and to some extent overcome DM1-associated defects. 29 In the IRES context, 2-aminobenzimidazole derivatives have also been shown to interact with the SLIIa structural element of the HCV-like IRES, 30,31 and to interfere with viral multiplication at low micromolar concentrations. 32,33 However, to the best of our knowledge, their potential to bind other types of IRES elements as well as cap-dependent translation has not been examined.…”

Functional analysis of Kaposi's sarcoma–associated herpesvirus vFLIP expression reveals a new mode of IRES-mediated translation

“…Much effort has been dedicated to the development of drugs that could impair IRES-mediated translation as a means of blocking viral replication, mainly focusing on the HCV IRES (Dibrov et al 2012(Dibrov et al , 2014. Recently it has been proposed that cap-independent expression of vFLIP could provide a mechanism to control the balance between vCyclin and vFLIP levels, thereby allowing vFLIP to suppress autophagy and inhibit senescence during latent infection (Leidal et al 2012).…”