Resurrection of a Viral Internal Ribosome Entry Site from a 700 Year Old Ancient Northwest Territories Cripavirus

Figures

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  Figure 1. (A) Secondary structure model of the ancient Northwest Territories Cripavirus (aNCV) intergenic region (IGR) (accession KJ938718.1) Pseudoknots (PK) I, II, III are indicated. Orange-colored nucleotides at Loop (L)1.1B, L3 denote sequences conserved in Type I IGR internal ribosome entry site (IRES), and blue-colored nucleotides at L1.1A indicate sequences conserved in Type II IGR IRES. Red-colored nucleotides at stem loop (SL) IV are unique in sequence in aNCV IGR IRES. Green-colored nucleotides represent IGR sequences outside the core IRES. The predicted start codon is GCU adjacent to the PKI domain. Numbering refers to the nucleotide position in the IGR as the genome of aNCV is not complete. (B) Alignment of Type I and II IGR IRESs with aNCV IGR. (C) Maximum-likelihood phylogeny of the Dicistroviridae Type I IRES nucleotide sequences. SH-like branch support values are indicated at nodes and the maximum-likelihood scale bar indicates average residues substitution per site.

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  Figure 2. Affinity of 80S-aNCV IGR IRES complexes. (A) Filter binding assays. [32P]-aNCV IGR IRES, cricket paralysis virus (CrPV) IGR IRES or mutant (ΔPKI/II/III) CrPV IGR IRES (0.5 nM) were incubated with increasing amounts of purified salt-washed 80S. The fractions bound were quantified by phosphor-imager analysis. (B) Competition assays. Quantification of radiolabeled 80S-CrPV IGR IRES (left) or 80S-aNCV IGR IRES (right) complex formation with increasing amounts of cold competitor RNAs (aNCV IGR IRES, CrPV IGR IRES or mutant (ΔPKI/II/III) CrPV IGR IRES). Shown are the averages ± standard deviation from at least three independent experiments.

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  Figure 3. aNCV IGR IRES bound to 80S is RNase I-resistant. Radiolabeled RNAs were incubated with 80S (0.6 μM) for 20 min before adding RNase I (1 U) for 1 hr. RNAs from RNase I treated/untreated reactions were TRIZOL-extracted and loaded onto urea-PAGE and visualized by phosphor-imager analysis. Shown is a representative gel from at least two independent experiments.

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  Figure 4. Toeprinting analysis of 80S-aNCV IRES complexes. (A) Schematic of IGR IRES PKI region. Mutations within the PKI region are annotated. (B) Primer extension analysis was performed on in vitro transcribed bicistronic RNAs containing the indicated wild-type or mutant CrPV IRES (left) or aNCV IRES or CrPV IRES (right) incubated in rabbit reticulocyte lysate (RRL) pretreated with cycloheximide (0.68 mg/mL). For the aNCV IRES, the sequencing ladder corresponds to nucleotides 279–331. The major toeprint is indicated at right, which is twenty nucleotides downstream of AUC289-91 of PKI given that the A is +1. (C) Primer extension analysis on in vitro transcribed bicistronic RNAs containing the indicated wild-type or mutant aNCV IRES incubated with purified ribosomes. Sequencing ladder corresponds to aNCV IRES nucleotides 269–319. The major toeprint is indicated at right, which is 13 nucleotides downstream of AUC289-91. Shown is a representative gel from at least three independent experiments.

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  Figure 5. In vitro translation assays in RRL. (A) Schematic of bicistronic reporter construct containing the IRES within the intergenic region. (B) In vitro-transcribed bicistronic reporter RNAs were incubated in rabbit reticulocyte lysate (RRL) for 60 min in the presence of [35S]-methionine/cysteine. The reactions were loaded on an SDS-PAGE gel, which was then dried and imaged by phosphor-imager analysis. (top) Integrity of the in vitro transcribed RNAs are shown. (bottom) Quantification of the radiolabeled Renilla (RLuc) and firefly (FLuc) luciferase proteins. ns, p > 0.05; **, p < 0.005. Shown are the averages ± standard deviation from at least three independent experiments.

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  Figure 6. Chimeric CrPV-aNCV clone is infectious in Drosophila S2 cells. (A) Schematic of the chimeric CrPV-aNCV clone replacing CrPV IGR IRES with that of the aNCV IRES. (B) In vitro translation of CrPV and CrPV-aNCV RNAs in Sf21 extracts. CrPV-ORF1-STOP contains a stop codon within the N-terminal ORF1, thus preventing expression of the non-structural proteins. Reactions were resolved by SDS-PAGE and visualized by autoradiography. (C) Immunoblotting of CrPV VP2 structural protein and 1A non-structural protein. (D) RT-PCR of viral negative-strand RNA from Drosophila S2 cells transfected with the indicated viral clone RNAs at 72 and 144 h after transfection. (E) Immunoblotting of CrPV 1A and VP2 proteins from lysates of Drosophila S2 cells infected with CrPV or CrPV-aNCV chimera (MOI 5) at the indicated h.p.i. Shown are representative gels from at least three independent experiments.