The Halastavi árva Virus Intergenic Region IRES Promotes Translation by the Simplest Possible Initiation Mechanism

Abstract

Dicistrovirus intergenic region internal ribosomal entry sites (IGR IRESs) do not require initiator tRNA, an AUG codon, or initiation factors and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A sites to P sites, bringing the first sense codon into the decoding center. Halastavi árva virus (HalV) contains an IGR that is related to previously described IGR IRESs but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By using in vitro reconstitution and cryoelectron microscopy (cryo-EM), we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.

Keywords: Cricket paralysis virus; Halastavi árva virus; IRES; SERBP1; SERPINE1 mRNA binding protein 1; dicistrovirus; intergenic region; internal ribosomal entry site; pseudoknot; ribosome.

Figure 1.. The Mechanism of Initiation on the HalV IGR IRES

(A) Schematic representation of various ribosomal complexes showing the relative positions of their toe-prints. (B) 48S complex formation on HalV IGR mRNA comprising a 5′-terminal stem (ΔG = −25.80 kcal/mol) followed by nucleotides 6211–7460 of the HalV genome in the presence of 40S subunits, Σaa-tRNA, and canonical eIFs, as indicated, assayed by toe-printing. The positions of AUG codons in the HalV IGR and assembled ribosomal complexes are indicated. (C) Direct binding of the HalV IGR mRNA to 80S ribosomes followed by elongation upon addition of eEF1H, eEF2, Σaa-tRNA, and cycloheximide, assayed by toeprinting. The positions of the A site and P site codons of the HalV IGR IRES are shown on the left. The positions of ribosomal complexes and the eEF2-mediated toe-print shift are indicated on the right. Separation of lanes by white lines indicates that they were juxtaposed from the same gel. (D) Association of ³²P-labeled HalV IGR-containing mRNA with individual 40S and 60S ribosomal subunits and 80S ribosomes, assayed by sucrose density gradient centrifugation. (E) Toe-printing analysis of ribosomal association of the HalV IGR IRES depending on the order of incubation of mRNA, 40S ribosomal subunits, and 60S ribosomal subunits. The position of IRES/80S complexes is indicated. (F) Ribosome-binding activity of the AUG_6397-6399→UAG stop codon HalV IGR mRNA mutant, assayed by toe-printing. The position of IRES/80S complexes is indicated. (G) The fidelity of reading frame selection on the HalV IGR IRES investigated by the ability of 80S/IRES complexes formed on GCC_6406-6408(Ala)→UCU(Ser), ACU_6409-6411(Thr)→UCU(Ser), and AUU_6412-6414(Ile)→UCU(Ser) HalV IGR variants and IGR mutants with UCU(Ser) placed in the +1 or +2 reading frame by insertion of one (G) or two (GC) nucleotides. between ACC_6403–6405 and UCU_6406–6408(Ser) codons to undergo one-cycle elongation in the presence of eEF1H, eEF2, and Ser-tRNA^Ser, assayed by toe-printing. The positions of IRES/80S binary complexes and 80S elongation complexes (80S ECs) are shown on the right. (H) Comparison of the eEF2 dependency of the A site accessibility in 80S ribosomal complexes assembled on HalV and CrPV IGR IRESs, assayed by RelE cleavage. Sites of RelE cleavages were determined by primer extension. The positions of P site codons, RelE cleavages, and IRES/80S control toe-prints are indicated. (I and J) Comparison of the eEF2 dependency of the A site accessibility in 80S ribosomal complexes formed on HalV and CrPV IGR IRESs by binding of eRF1 and eRF3 to 80S ribosomes assembled on (I) HalV IRES-STOP(UAA) and (J) CrPV IRES-STOP(UAA) mutant mRNAs, assayed by toe-printing. The positions of ribosomal complexes and P site and stop codons are indicated. (K) The ability of HalV IGR IRES to form elongation-competent complexes with insect (Spodoptera frugiperda) 80S ribosomes, assayed by toe-printing. The positions of the A site codon and ribosomal complexes are indicated. (B, C, and G–K) Lanes C, T, A, and G depict CrPV or HalV sequences, as indicated.

Figure 2.. The Influence of SERBP1 on Initiation on the HalV IRES

(A) Translation in RRL driven by HalV 5′ UTR and IGR IRESs, depending on addition of 40S and 60S subunits with or without their preincubation with mRNA. (B) Protein composition of 80S ribosomes reconstituted from individual purified 40S and 60S subunits, and native 80S ribosomes purified from RRL, assayed by SDS-PAGE followed by SYPRO staining. (C) The influence of SERBP1 with/without eEF2 on ribosomal binding of the HalV IGR IRES, assayed by toe-printing. The positions of the A site codon and ribosomal complexes are indicated. Separation of lanes by white lines indicates that they were juxtaposed from the same gel. (D) Comparison of the binding of the HalV IGR IRES to reconstituted and native 80S ribosomes, assayed by toe-printing. The positions of the A site codon and ribosomal complexes are indicated. (C and D) Lanes C, T, A, and G depict HalV sequence. (E) The influence of SERBP1 with/without eEF2 on ribosomal binding of CrPV IGR IRES, assayed by toe-printing. The positions of toe-prints corresponding to IRES/80S complexes are indicated.

Figure 3.. Structure and Mutational Analysis of the HalV IGR IRES

(A) Model of the HalV IGR IRES and the adjacent 3'-terminal region of ORF1, derived on the basis of computational analysis (see STAR Methods) and chemical/ enzymatic foot-printing (Figures S1A and S1B). It is annotated to show nucleotides at 20 nt intervals, IRES domains and secondary structural elements (based on the nomenclature proposed by Costantino and Kieft (2005)), the ORF1 stop codon (UAA₆₂₇₆) and the ORF2 A site codon (GCC₆₄₀₆) (both boxed blue). Arrows indicate the 5′ borders of truncated HalV IGR mRNAs used in experiments to assay IRES activity in ribosome binding (C and D). (B) Model of the CrPV IGR IRES, showing IRES domains and secondary structure elements, nucleotides that interact with ribosomal proteins and elements of 18Sand 28S rRNAs, and conserved motifs in the L1.1 loop in dicistrovirus IGR IRESs (Nishiyama et al., 2003; Pfingsten et al., 2006). (C and D) The 5′-terminal border of the HalV IGR IRES assayed by toe-printing of IRES/80S complex formation. The position of IRES/80S binary complexes is indicated. Lanes C, T, A, and G depict HalV sequence. (E and F) Analysis of the influence of disruptive and compensatory substitutions in helical elements of domain 3/PKI (E) and domain 1/PKII (F) on the HalV IGR IRES’s ribosome-binding (E and F) and elongation (E) activity, analyzed by toe-printing. The positions of IRES/80S binary complexes and 80S ECs are indicated. (G) Analysis of the influence of substitutions in single-stranded elements of the HalV IGR IRES on its ribosome-binding activity, analyzed by toe-printing. The position of IRES/80S binary complexes is indicated. (F and G) Separation of lanes by white lines indicates that they were juxtaposed from the same gel.

Figure 4.. Overview of the HalV IGR IRES Bound to the O. cuniculus 80S Ribosome

(A) Superimposition of the 60S subunit from the unrotated and rotated complexes, to emphasize 40S and IRES movement. (B) Cryo-EM map of the unrotated complex. The 40S is semi-transparent to reveal the path of the IRES. (C) The 3D-based secondary structure diagrams of the IGR IRES from HalV (top) and CrPV (bottom). Color coding by domain according to functional role. Residueconservation: >80%, red; 60%–80%, blue (within a set of 19 IGR IRES sequences [see Figure S2]). Inset, classical representation of a secondary structure diagram for the CrPV IRES. (D) Comparison of the overall structures of HalV and CrPV. Color coding as in (C).

Figure 5.. Flexibility of the HalV IRES Is Key to Its Function

(A) Compression of the IRES in its central region during rotation. Superimposition of filtered density maps (Gaussian filter 1.5) of the unrotated and rotated states of the 80S-bound IRES. The circle indicates the region where additional bulging density is observed in the rotated complex. (B) Superimposition as in (A) of the IRES 3D models (color coding as in A). Vectors help visualize the direction of the movement as well as its amplitude. Vectors were calculated by measuring the distance between phosphate atoms. (C) The central region of the IRES is the most dynamic. Color coding of the unrotated IRES by atomic displacement parameter (ADP). (D) Mutations that impair function cluster to regions interacting with ribosomal proteins and to the central region. Color coding of the IRES according to the effect of mutations on its function. Percentage of activity in comparison with wild-type RNA.

Figure 6.. Contacts between the HalV IRES and Ribosomal Proteins

Overview of a cross section of the ribosome showing the 80S-bound HalV IGR IRES in the unrotated state. The exposed section is indicated on the small 80S/HalV IRES schema on the right by a red rectangle. (A) Blow-up figures on the HalV IRES interaction with the 80S ribosome in the unrotated state. (B) Interactions between PKI and 18S rRNA in the unrotated state. (C) View as in (A) for the rotated state. Note the missing interaction between the HalV IRES and uS11 in this state. (D) View as in (B) for the rotated state. The exposed area is indicated on the small 80S/HalV IRES schema below (B) and (D) by a black circle. IRES nucleotides,which interact with ribosomal proteins and 18S rRNA and which were shown by mutational analysis to be important for IRES function (Figures 3G and 7A), are marked with asterisks. Note that the PK1 region in the rotated state is more flexible than in the unrotated state, as demonstrated by the scanter local densities and resolution compared with the unrotated state. Color coding for all panels: 40S, dark/light yellow; 60S, dark/light blue; HalV, dark/light green; uL1, magenta; uS11, yellow; uS7, red; uL5, teal; uS3, light blue; uS9, purple.

Figure 7.. The P-Site-Stabilizing Interaction of PKI and Activities of Hybrid HalV/CrPV IGR IRESs

(A) Left: model of the HalV IGR PKI structure, showing nucleotides targeted for substitution to assay a stabilizing interaction of PKI in the P site. Right: binding of 80S ribosomes to the indicated HalV IGR IRES mutants, assayed by toe-printing. (B, C, E, and F) Ribosomal binding, one-cycle elongation and release factor binding on WT and hybrid HalV/CrPV IGR IRESs (shown schematically in each panel) in the presence of the indicated translational components, assayed by toe-printing. The positions of ribosomal complexes are shown. (D) Ribosome-binding activity of the L2 deletion/insertion HalV IGR IRES mutants, assayed by toe-printing. Separation of lanes by white lines indicates that they were juxtaposed from the same gel. (A–F) The positions of ribosomal complexes are indicated. (A, B, and F) Lanes C, T, A, and G depict CrPV or HalV sequences.