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, 38 (21), e102226

The Israeli Acute Paralysis Virus IRES Captures Host Ribosomes by Mimicking a Ribosomal State With Hybrid tRNAs

The Israeli Acute Paralysis Virus IRES Captures Host Ribosomes by Mimicking a Ribosomal State With Hybrid tRNAs

Francisco Acosta-Reyes et al. EMBO J.

Abstract

Colony collapse disorder (CCD) is a multi-faceted syndrome decimating bee populations worldwide, and a group of viruses of the widely distributed Dicistroviridae family have been identified as a causing agent of CCD. This family of viruses employs non-coding RNA sequences, called internal ribosomal entry sites (IRESs), to precisely exploit the host machinery for viral protein production. Using single-particle cryo-electron microscopy (cryo-EM), we have characterized how the IRES of Israeli acute paralysis virus (IAPV) intergenic region captures and redirects translating ribosomes toward viral RNA messages. We reconstituted two in vitro reactions targeting a pre-translocation and a post-translocation state of the IAPV-IRES in the ribosome, allowing us to identify six structures using image processing classification methods. From these, we reconstructed the trajectory of IAPV-IRES from the early small subunit recruitment to the final post-translocated state in the ribosome. An early commitment of IRES/ribosome complexes for global pre-translocation mimicry explains the high efficiency observed for this IRES. Efforts directed toward fighting CCD by targeting the IAPV-IRES using RNA-interference technology are underway, and the structural framework presented here may assist in further refining these approaches.

Keywords: Israeli acute paralysis virus; internal ribosomal entry sites; ribosome; translation.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. IAPV‐IRES secondary structure, experimental set‐up, and cryo‐EM image processing workflow

IAPV‐IRES diagram colored according to secondary structure motifs. Bottom, a closer view of the IAPV‐IRES PKI highlighting its sequence, with base pairs indicated as well as the variable loop region (VLR) and stem loop III (SL‐III).

Sucrose gradient UV profile of 60S/40S/IAPV‐IRES reaction mixture after an overnight run. The peaks corresponding to 80S and 40S were used for RNA extraction and UREA‐PAGE shown in the inset.

Representative cryo‐EM image where roughly half of the particles correspond to 40S (blue) and the other half to 80S (orange).

Two classes with robust density for the IAPV‐IRES were found in the 40S group.

After classification, three classes with clear IAPV‐IRES density and small differences in the conformation of the 40S were found in the 80S group.

Figure 2
Figure 2. Structure of the IAPV‐IRES in complex with the 40S ribosomal subunit

Overview of the mammalian 40S in complex with IAPV‐IRES. Left, cryo‐EM final post‐processed map of class 1 with 40S colored yellow and IAPV‐IRES maroon. Right, corresponding final refined model with IAPV‐IRES colored according to Fig 1A.

Ribbon diagram of the 40S colored by pairwise root‐mean‐square deviation displacements observed between the two IAPV‐IRES/40S classes. The different position of the 40S head between both classes is a composition of swiveling and tilt movements (indicated by arrows in orthogonal views).

Close‐up view of the ribosomal sites of the 40S for IAPV‐IRES/40S class 1 showing cryo‐EM unsharpened cryo‐EM density.

Sequence of the PKI three‐way helical junction.

Unsharpened cryo‐EM density for the PKI region of the IAPV‐IRES in class 1 with the SL‐III and the tRNA/mRNA mimicking domain indicated.

Figure 3
Figure 3. IAPV‐IRES conformation in the context of a 40S interaction

Superposition of IAPV‐IRES models corresponding to IAPV‐IRES/40S class 1 and class 2 after alignments excluding the IRES and the 40S head. A similar conformation can be observed with distinctive relative orientation with respect to the 40S body. The movement characteristic of the 40S head is indicated by arrows in orthogonal views.

Ribbon diagram of the IAPV‐IRES colored by pairwise root‐mean‐square deviation displacements observed between the two IAPV‐IRES/40S classes. The ASL/mRNA‐like regions of the PKI and well as the SL‐IV show the lowest degree of displacement (blue), whereas the apical part of SL‐III and the L1.1 the highest (red).

Superposition of the IAPV‐IRES with tRNAs in different configurations indicated at the bottom. IAPV‐IRES is depicted as ribbons colored according to the secondary structure elements, and tRNAs are represented as gray ribbons. Alignments of the models were computed with the 40S body, excluding from the computation the ligands (IRES/tRNAs) and the 40S head.

Detailed view of the refined model for IAPV‐IRES/40S class 1 inserted in the post‐processed cryo‐EM density focused on the decoding center of the 40S. PKI of IAPV‐IRES is depicted green and 18S rRNA yellow.

Close‐up view of the refined model for IAPV‐IRES/40S class 1 inserted in the post‐processed cryo‐EM density focused on the SL‐IV of the IAPV‐IRES (depicted blue).

Figure 4
Figure 4. SL‐III of IAPV‐IRES engages novel sites of the 60S ribosomal subunit

Overall view of the IAPV‐IRES/80S complex class 1 with 60S represented as cyan ribbons, the 40S as yellow ribbons, and the IAPV‐IRES represented as solid Van der Waals surface colored by secondary structure motifs.

Close‐up view of the intersubunit space with the IAPV‐IRES depicted as cartoons colored as in (A) inserted in the unsharpened cryo‐EM density.

Zoomed view of the A site finger in interacting distance with the SL‐III (green). The apical loop of SL‐III reaches deep into the 60S contacting the ribosomal protein uL16.

Superposition of the IAPV‐IRES in complex with 80S (class 1) with tRNAs in different configurations indicated at the bottom. Alignments of the models were computed with the 40S body, excluding from the computation the ligands (IRES/tRNAs) and the 40S head. IAPV‐IRES PKI component SL‐III/ASL‐like domain populates a space of the intersubunit space similar to a A/P‐tRNA. IAPV‐IRES PKIII (red) mimics the elbow region of a hybrid P/E‐tRNA.

Figure 5
Figure 5. The IAPV‐IRES restricts the small subunit rotational dynamics in a pre‐translocation complex with 80S

Ribbon diagram of IAPV‐IRES/80S complex viewed from the 40S colored by pairwise root‐mean‐square deviation displacements observed between the classes indicated on the left. Class 1 is unrotated, while classes 2 and 3 exhibit a small rotational movement of the 40S.

Top, general overview of the non‐rotated IAPV‐IRES/80S class 1 structure. IAPV‐IRES is depicted as solid Van Der Waals surface colored according to the secondary structure motifs. The PKI (green) is solidly anchored to the A site. Bottom, close‐up view of the IAPV‐IRES PKI inserted in unsharpened map.

Zoomed view of A3760, a nucleotide belonging to the helix 69 (H69) of the 28S rRNA interacting with PKI. Final refined model inserted in the post‐processed map is shown.

This interaction is not disrupted along the small fluctuations of the 40S. An apical view along the axis of the PKI of a superposition of class 1 versus class 2 shows the IRES displacements are minimal and are followed by the H69 which constantly interacts with the IRES.

Figure 6
Figure 6. Visualization of IAPV‐IRES in a post‐translocated state in the ribosome

Biochemical strategy employed to trap a post‐translocated state of IAPV‐IRES in the ribosome.

General view of the IAPV‐IRES in a post‐translocated state on the ribosome: 60S depicted as blue ribbons, 40S as yellow ribbons, IAPV‐IRES represented as solid Van Der Waals surface colored according to secondary structure elements described in Fig 1A, and eRF1* depicted orange.

Close‐up view of the SL‐III inserted in the experimental unsharpened cryo‐EM density. On the right, refined model with residues from the 60S (blue) in interacting distance with the SL‐III (green) indicated.

Comparison of the final refined model for IAPV‐IRES colored according to the secondary structure described in Fig 1A with canonical tRNAs (PDBID: 4V5D) in the pre‐translocated state (left) and after translocation (right).

Left, overall top view of the intersubunit space of the 40S for the post‐translocated state. Inset, close‐up view of the 40S tRNA binding sites where it can be appreciated the insertion of PKI of the IAPV‐IRES in the P site, projecting the VLR toward the E site. The elements of the 18S rRNA forming the “P site gate” are indicated by solid arrows. On the right, equivalent view for canonical tRNAs.

Figure 7
Figure 7. Type IV IRES families exploit different pre‐translocation features of canonical translation for ribosome hijacking
(Top), The Cripavirus family of type IV IRESs is able to capture free 40S subunits and engage them on a pre‐translocation complex by recruiting 60S. This complex is extremely dynamic, with the 40S alternating between non‐rotated and rotated configurations with respect to the 60S subunit. IRESs belonging to this family, exemplified by the CrPV‐IRES, recruit elongation factors by mimicking a rotated stated of the ribosome with tRNAS. (Bottom), The Aparavirus family of IRESs follows a similar pathway in order to assemble a pre‐translocation complex; however, specific structural components of this family allow for additional contacts with the 60S, limiting the rotational freedom of the 40S. Elongation factor engagement and thus effective ribosome hijacking are accomplished by mimicking a ribosome state with tRNAs in hybrid configurations.

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