HCV IRES interacts with the 18S rRNA to activate the 40S ribosome for subsequent steps of translation initiation

Abstract

Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES-rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.

Figure 1.

The HCV IRES constructs. (A) The HCV IRES secondary structure (6,21,22). Domains (large roman numbers) and subdomains (small roman numbers) are indicated, the initiator AUG codon is underlined, and the ORF is marked. (B) The cartoons representing simplified secondary structures of the HCV IRES constructs used in the study; their abbreviations are given in the text. (C) Isotherms of binding of IRES_aug (circles) and IRES_augΔDII (squares) to 40S ribosomal subunits at 25°C. (D) Isotherms of binding of IRES_fl (circles) and IRES_flΔDII (squares) to the 40S ribosomal subunits at 37°C.

Figure 2.

Direct hydroxyl radical probing of 18S rRNA from human 40S ribosomal subunits in the complexes with IRES_aug or IRES_augΔDII. Reverse transcription analyses of the 18S rRNA regions 1070–1130 (A), 1600–1660 (B), 495–554 (C) and 522–558 (D) with the use of respective ³²P-labelled primers are presented. Sequencing lanes are indicated as A, C, G and U. Other lanes correspond to 18S rRNA isolated from free 40S subunits either untreated (40S) or treated with hydroxyl radicals (marked with OH^•) and from complexes of 40S subunits with IRES_aug (40S-IRES_aug) or IRES_augΔDII (40S-IRES_augΔDII) treated with hydroxyl radicals. Positions of reverse transcription stops on the 18S rRNA nucleotides 3′ to the nucleotides, whose ribose moieties were subjected to hydroxyl radical attack, are indicated on the right. (E) Cartoon of the human 18S rRNA secondary structure [adapted from the structures reported in (24,25)]. Positions of IRES-dependent protections and enhancements are shown by filled and open circles, respectively.

Figure 3.

Interaction between the HCV IRES subdomain IIId apical loop and the loop of 18S rRNA ES7. (A) Comparison of the cryo-EM derived structure of the 40S ribosomal subunit bound with the HCV IRES (8) (on the top) and the crystal structure of the 40S ribosomal subunit (24) (on the bottom) taken in the same orientations. Positions of the 18S rRNA h26 apical loop and the IRES subdomain IIId are indicated. (B) Reverse transcription analysis of the 18S rRNA h26 from free human 40S ribosomal subunits either untreated (lanes 40S, − DMS) or treated with DMS (40S, + DMS) and from complex of 40S subunits with IRES_aug treated with DMS (40S-IRES_aug, + DMS). A, C, G and U are sequencing lanes. Positions of reverse transcription stops on 18S rRNA nucleotides 3′ to the nucleotides protected from modification by IRES_aug are marked on the left. (C) Secondary structures of the human 18S rRNA h26 upper part (on the top) where nucleotides protected by IRES_aug from DMS modification (circles) are indicated, and of the HCV IRES subdomain IIId (on bottom) with marks corresponding to the conserved GGG triplet (encircled) protected by 40S subunits from RNase digestion (20,26).

Figure 4.

Direct hydroxyl radical probing of 18S rRNA region G1639 in human 40S ribosomal subunits complexed with HCV IRES constructs. (A) Reverse transcription analysis of the 18S rRNA isolated from free 40S subunits either untreated (40S) or treated with hydroxyl radicals (marked with OH^•) and from complexes of 40S subunits with IRES_fl (40S-IRES_fl), IRES_aug (40S-IRES_aug) or IRES_flΔDII (40S-IRES_flΔDII) treated with hydroxyl radicals. (B) Reverse transcription analysis of the 18S rRNA isolated from free 40S subunits either untreated (40S) or treated with hydroxyl radicals (marked with OH^•) and from complexes of 40S subunits with IRES_aug (40S-IRES_aug), IRES_ΔDIV (40S-IRES_ΔDIV) or IRES_uuu (40S-IRES_uuu) treated with hydroxyl radicals. Positions of reverse transcription stops on 18S rRNA nucleotides 3′ to the nucleotides, whose ribose moieties were subjected to hydroxyl radical attack, are indicated on the right in panels (A) and (B).

Figure 5.

A hypothetical model of the process of HCV IRES adaptation to the 40S subunit up to the 48S complex formation. Positions of rpS5 (green), ES7 (indicated by arrow) and G1639 (red) are designated on the simplified view of the eukaryotic 40S ribosomal subunit, in accordance with (27). States of the process are indicated by roman numbers and described in the text. The regions where key events occur are encircled with red.