Initiation of translation on nedicistrovirus and related intergenic region IRESs by their factor-independent binding to the P site of 80S ribosomes

Figures

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  FIGURE 1.. Schematic models of different types of IGR IRES. Models of (A) a type 6a IRES (e.g., cricket paralysis virus), (B) a type 6b IRES (e.g., Taura syndrome virus), (C) a type 6c IRES (e.g., Halastavi árva virus), and (D) a type 6d IRES (e.g., nedicistrovirus), labeled to indicate domain 1 (PKII) colored blue, domain 2 (PKIII) colored red and domain 3 (PKI) colored green, stem–loops SLIII, SLIV, and SLV, and the L1.1a loop.

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  FIGURE 2.. Sequence alignments and structural models of NediV and APLV1 IGR IRESs. (A) Aligned sequences from nedicistrovirus (NediV), Antarctic picorna-like virus 1 (APLV1), a viral TSA fragment from Pallasea cancelloides (Pallasea), Changjiang picorna-like virus 9 (Changjiang9), Beihai picorna-like virus 78 (Beihai78), Gingko biloba dicistrovirus (Gingko), Picornavirales sp. isolates R35-k141_316374 (Pico R35) and s64-k141_2464283 (Pico s64), and Sanxia picorna-like virus 12 (Sanxia12), annotated to show nucleotide numbers, structural elements (as in panels B,C), the ORF2 initiation codon, conserved sequence motifs (blue), base-paired nucleotides (bold), unpaired residues (light gray or light blue), and conserved nucleotides (indicated by asterisks). (B,C) Models of (B) NediV and (C) APLV1 IRESs. Nucleotides are numbered at 20-nt intervals. Domains 1 (PKII), 2 (PKIII), and 3 (PKI) are colored red, blue, and black, respectively. The 5′ borders of truncated IGR mRNAs used to define the IRES 5′ border (see Fig. 4A,B) are indicated by arrows. The NediV codon GCU1493–5 and the APLV1 codon GCU5604–6 that predominantly occupy the A site on initial binding of ribosomes, and the NediV UAG1330–2…

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  FIGURE 3.. The mechanism of initiation on type 6d IRESs. (A,B) Assembly of ribosomes from 40S and 60S subunits on (A) NediV, (B) APLV1 and (A,B) CrPV and HalV IRESs, assayed by toeprinting. The NediV CCC1490–2 and APLV1 CUC5601–3 triplets that precede ORF2 are indicated on the left and the positions of toeprints relative to these codons are indicated on the right of panels A–K. (A) The +15–17 nt toeprints on the right correspond to binding of NediV CCC1490–2 in the P site of IRES-bound ribosomes; the +19–20 toeprints are discussed in the text. (B) The +16–17 nt toeprints on the right correspond to binding of APLV1 CUC5601–3 in the P site of IRES-bound ribosomes, analogously to the toeprints +14–15 nt from CrPV CCU6214–6 and +15–17 nt from HalV ACC6403–5. (C) Assembly of S. frugiperda ribosomes on the NediV IRES, assayed by toeprinting. (D–F) Toeprinting analysis of ribosome assembly and subsequent elongation on (D) APLV1 wt, (E) NediV wt, and (F) APLV1[GCU-UCU] mutant mRNAs incubated with ribosomal subunits with (D,E) or without (F) canonical eIFs and Met-tRNAiMet and with (D,E) eEF1H/2, ∑aa-tRNA and cycloheximide (CHX) or (F) eEF1H/2 and Ser-tRNASer. The shifts in toeprints (D) caused by…

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  FIGURE 4.. Inhibition of NediV IGR IRES function by SERBP1/eEF2. (A,B) Translation of 5′-stem-NediV IRES mRNAs in RRL with or without preincubation with purified 40S subunits or 80S ribosomes. (A) FLuc and CrPV dual Luc mRNAs are positive controls. The FLuc and NediV ORF2 translation products are indicated on the right and molecular weight markers (kDa) on the left. (B) Translation of 5′-stem-NediV IRES + 5 kDa and mutant versions of 5′-stem-NediV IRES mRNA with substitutions in IRES domain 1 (N6), domain 2 (N33), and domain 3 (N59 and N61), after preincubation with 80S ribosomes. FLuc mRNA was a positive control. The FLuc, NediV ORF2, and NediV-(ORF2 + 5 kDa) translation products are indicated on the right and molecular weight markers (kDa) on the left. (C) Influence of SERBP1 and eEF2 on binding of ribosomes to the NediV IRES, assayed by toeprinting. The positions of +16–+17 nt and +19 nt toeprints are shown on the right. Lanes C, T, A, G depict the NediV sequence. (D,E) Influence of SERBP1 and eEF2 on ribosomal complex formation on (D) APLV1 and (E) CrPV IRESs incubated with SERBP1, eEF2, and ribosomal subunits or native ribosomes, as indicated. The positions of the P-site codon and of…

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  FIGURE 5.. Mutational analysis of domain 1/PKII. (A,C) Toeprinting analysis of the influence of 5′-terminal deletions of (A) the NediV IRES and (B) the APLV1 IRES on their ability to support binding of ribosomes and subsequent elongation upon inclusion of the indicated components. The positions of the codons that occur immediately upstream of ORF2 coding sequences are indicated on the left, and full-length cDNAs and toeprints from ribosomal complexes before and after elongation are indicated on the right. Lanes C, T, A, G depict (A) NediV and (C) APLV1 sequences. (B) Association of 32P-labeled wt and mutant NediV IGR-containing mRNAs with 80S ribosomes, assayed by sucrose density gradient centrifugation. Mutant mRNAs had substitutions in domain 1 (N6, N13) or in domain 3 (N57). Sedimentation was from right to left, and the position of 80S complexes is indicated. Fractions from the upper part of the sucrose gradient have been omitted for greater clarity. (D,E) The influence of disruptive and compensatory substitutions in P1.1, P1.2, and P1.3 helices and the L1.1 loop of (D) APLV1 and (E) NediV IRESs, as indicated on the models of IRES domain 1. IRES activity in ribosomal binding was assayed…

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  FIGURE 6.. Mutational analysis of domain 2/PKIII. (A) Influence of APLV1 PKIII deletion on 80S:IRES complex formation, assayed by toeprinting. The influence of disruptive or compensatory substitutions in domain 2 of (B–H) the APLV1 IRES and (I–Q) the NediV IRES on binding of ribosomes, assayed by toeprinting. (A–H) Separation of lanes by white lines indicates that they were juxtaposed from the same gel. Positions of 80S:IRES complexes are indicated on the left (A) or right (C–Q).

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  FIGURE 7.. Mutational analysis of domain 3/PKI. The influence of disruptive or compensatory substitutions in domain 3 of (A–G,N) the NediV IRES and (H–M,O) the APLV1 IRES on binding of ribosomes, assayed by toeprinting. Separation of lanes by white lines indicates that they were juxtaposed from the same gel. Positions of 80S:IRES complexes are indicated on the right. Summary of the effects of substitutions in (N) NediV and (O) APLV1 IRESs. Models of these IRESs are annotated to show mutations that (a) have no effect, (b) cause loss of function that is compensated by second-site mutations, and (c) impair function or lead to loss of function that is not compensated by second-site mutations, as indicated in the inset key.

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  FIGURE 8.. 80S complex formation on chimeric NediV/APLV1 and NediV/CrPV IRESs. (A) Schematic models of hybrid IRESs. Elements derived from NediV, APLV1, and CrPV IRESs are colored light blue, black, and red, respectively. (B) Binding of ribosomes to NediV wt and NediV/APLV1 chimeric IRESs, assayed by toeprinting. Separation of lanes by white lines indicates that they were juxtaposed from the same gel. (C) Binding of 40S or 40S and 60S subunits to NediV wt and NediV/CrPV chimeric IRESs, followed by elongation on inclusion of eEF1H/2H, ∑aa-tRNA, and cycloheximide (CHX), assayed by toeprinting. (B,C) Positions of the P site codon and of bound ribosomal complexes are indicated. Lanes C, T, A, G depict NediV sequence.

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  Anna Miścicka.

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  Kristen Lu.