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. 2000 Mar;74(5):2219-26.
doi: 10.1128/jvi.74.5.2219-2226.2000.

Interactions of viral protein 3CD and poly(rC) binding protein with the 5' untranslated region of the poliovirus genome

A V Gamarnik  1 , R Andino
Affiliations

Interactions of viral protein 3CD and poly(rC) binding protein with the 5' untranslated region of the poliovirus genome

A V Gamarnik et al. J Virol. 2000 Mar.

Abstract

The poly(rC) binding protein (PCBP) is a cellular protein required for poliovirus replication. PCBP specifically interacts with two domains of the poliovirus 5' untranslated region (5'UTR), the 5' cloverleaf structure, and the stem-loop IV of the internal ribosome entry site (IRES). Using footprinting analysis and site-directed mutagenesis, we have mapped the RNA binding site for this cellular protein within the stem-loop IV domain. A C-rich sequence in a loop at the top of this large domain is required for PCBP binding and is crucial for viral translation. PCBP binds to stem-loop IV RNA with six-times-higher affinity than to the 5' cloverleaf structure. However, the binding of the viral protein 3CD (precursor of the viral protease 3C and the viral polymerase 3D) to the cloverleaf RNA dramatically increases the affinity of PCBP for this RNA element. The viral protein 3CD binds to the cloverleaf RNA but does not interact directly with stem-loop IV nor with other RNA elements of the viral IRES. Our results indicate that the interactions of PCBP with the poliovirus 5'UTR are modulated by the viral protein 3CD.

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Figures

FIG. 1
FIG. 1
PCBP-RNA interactions within stem-loop IV of the poliovirus IRES. (A) Footprinting analysis reveals that PCBP protects several regions of the large stem-loop IV from RNase digestion. The viral 5′UTR was treated with RNase T1 (lanes 5 to 8) or RNase T2 (lanes 9 to 12). Lane 13 corresponds to untreated RNA. RNase was added in the presence of bovine serum albumin (−) or PCBP protein, as indicated at the top of each lane. Primer extension was performed by using a primer complementary to nucleotides 368 to 408. Lanes labeled C, T, A, and G correspond to dideoxy sequencing lanes. Brackets indicate PCBP protected regions (a, b, and c). The numbers on the left indicate the nucleotide position of the viral genome in poliovirus type 1. (B) Predicted secondary structure of stem-loop IV of the poliovirus type 1 genome. PCBP protected regions as determined by footprinting analysis in panel A are indicated by black lines (loop a, loop b, and bulge c).
FIG. 2
FIG. 2
Effect of mutations within stem-loop IV on PCBP binding and viral translation. (A) Position and sequence of mutations within the predicted secondary structure of stem-loop IV. The C-rich loop a and loop b are indicated with their wild-type and mutated sequences: SL IV-23, SL IV-298, and SL IV-332. (B) RNA mobility shift analysis showing the effect of mutations within stem-loop IV probe on PCBP binding. RNA mobility shift experiments were performed with four different probes: wild-type stem-loop IV (lanes 1 to 5), SL IV-23 (lanes 6 to 10), SL IV-298 (lanes 11 to 15), and SL IV-332 (lanes 16 to 20). Each probe was incubated with buffer (−) or increasing concentrations of cellular PCBP (0.2 ng to 0.1 μg), as indicated at the top of the panel. Position of specific complexes I and II and the stem-loop IV probe (Free Probe) are indicated by arrows. (C) Binding affinity of PCBP to wild-type and mutated stem-loop IV RNAs. The fraction of radiolabeled probe bound (Θ) determined by mobility shift assays is plotted against PCBP concentration. (D) Translation efficiencies of wild type and stem-loop IV mutants in HeLa cells. At the top, a schematic representation of the poliovirus replicon carrying the luciferase reporter gene in place of the capsid proteins. The arrow indicates a recognition site for cleavage by the viral 2A protease. At the bottom, the translation of wild type and stem-loop IV mutants (SL IV-23, SL IV-298, and SL IV-332) is measured as luciferase activity and plotted as a function of the time after RNA transfections into HeLa cells.
FIG. 2
FIG. 2
Effect of mutations within stem-loop IV on PCBP binding and viral translation. (A) Position and sequence of mutations within the predicted secondary structure of stem-loop IV. The C-rich loop a and loop b are indicated with their wild-type and mutated sequences: SL IV-23, SL IV-298, and SL IV-332. (B) RNA mobility shift analysis showing the effect of mutations within stem-loop IV probe on PCBP binding. RNA mobility shift experiments were performed with four different probes: wild-type stem-loop IV (lanes 1 to 5), SL IV-23 (lanes 6 to 10), SL IV-298 (lanes 11 to 15), and SL IV-332 (lanes 16 to 20). Each probe was incubated with buffer (−) or increasing concentrations of cellular PCBP (0.2 ng to 0.1 μg), as indicated at the top of the panel. Position of specific complexes I and II and the stem-loop IV probe (Free Probe) are indicated by arrows. (C) Binding affinity of PCBP to wild-type and mutated stem-loop IV RNAs. The fraction of radiolabeled probe bound (Θ) determined by mobility shift assays is plotted against PCBP concentration. (D) Translation efficiencies of wild type and stem-loop IV mutants in HeLa cells. At the top, a schematic representation of the poliovirus replicon carrying the luciferase reporter gene in place of the capsid proteins. The arrow indicates a recognition site for cleavage by the viral 2A protease. At the bottom, the translation of wild type and stem-loop IV mutants (SL IV-23, SL IV-298, and SL IV-332) is measured as luciferase activity and plotted as a function of the time after RNA transfections into HeLa cells.
FIG. 3
FIG. 3
PCBP dissociates from stem-loop IV in the presence of cloverleaf RNA and 3CD. Uniformly labeled stem-loop IV (5 ng) was incubated with buffer, unlabeled cloverleaf RNA (500 ng, lane 2), S10 HeLa proteins (20 μg), and decreasing concentrations of unlabeled cloverleaf (CL) RNA (500, 50, and 5 ng) (lanes 4 to 6 and lanes 7 to 9). Binding reactions were performed in the absence (lanes 3 to 6) or in the presence (lanes 7 to 10) of 0.5 μg of recombinant 3CD. The electrophoretic mobility of the two RNP complexes formed between PCBP and stem-loop IV (complex I and complex II) and the free stem-loop IV RNA (Free Probe) is indicated on the left.
FIG. 4
FIG. 4
Effect of 3CD on the affinities of PCBP for stem-loop IV and the cloverleaf. The fraction of radiolabeled probe bound (Θ) for stem-loop IV (A) and cloverleaf (B) is plotted against PCBP concentration. The bands corresponding to free and bound probes in the mobility shift assays were quantified in a PhosphorImager. The experiment was performed in the absence (■) or in the presence (□) of 0.5 μg of recombinant 3CD protein.
FIG. 5
FIG. 5
Footprinting analysis reveals that 3CD induces dissociation of PCBP from stem-loop IV. The footprinting analysis was performed as described for Fig. 1A. The viral 5′UTR was treated with RNase T1 (lanes 1 to 12) or RNase T2 (lanes 13 to 24). RNase was added in the presence of bovine serum albumin (−), 3CD, PCBP, or 3CD plus PCBP proteins, as indicated on the top of each lane. The numbers on the left indicate the nucleotide position in the poliovirus type 1 genome.
FIG. 6
FIG. 6
Interaction of 3CD with the 5′UTR of the poliovirus genome. (A) Schematic representation of the poliovirus 5′UTR. Predicted stem-loop I (cloverleaf) and stem-loops II to VI (IRES) are indicated. AUG at position 743 represents the authentic poliovirus initiation codon. (B) The viral protein 3CD does not interact with the poliovirus IRES. Western blot analysis with anti-3CD antibodies shows the viral protein precipitated by immobilized RNAs. The biotinylated RNA molecules used, 5′UTR, IRES, and GFP (GFP mRNA), are indicated on the top. In lane 4, no RNA was included in the reaction. The electrophoretic mobility of 3CD is indicated on the left. (C) Mobility shift competition experiments. Uniformly labeled cloverleaf RNA (1 ng, 30,000 cpm) was incubated with purified recombinant 3CD (0.5 μg) and with each of the six predicted stem-loops of the poliovirus 5′UTR as unlabeled competitors. Decreasing amounts of competitor RNAs (500, 50, and 1 ng) were used as indicated at the top of the gel with triangles. The mobility of the RNP complex corresponding to cloverleaf-3CD (RNP-b), and the free cloverleaf (Free Probe) is indicated on the left.
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