Characterization of Mutational Tolerance of a Viral RNA–Protein Interaction

Figure 1. Schematic representation of the poliovirus genome, its replicative cis-elements, and formation of the oriL/3CD complex. The positions of only 3C- and 3D-coding sequences are shown in the viral polyprotein reading frame (rectangle). Locations of the major replicative cis-elements (oriL, cre, and oriR) are indicated.

Figure 2. Generation of plasmids encoding viral genomes with randomized segments (A) or distinct mutations (B).

Figure 3. SDS PAGE profiles of the engineered mutant 3CD proteins.

Figure 4. Plaque phenotypes of the engineered viruses. Fifty ng of transcripts were used for the transfection with wild type genomes in all these experiments. (A,B) Altered tripeptide TGK. Twenty to 50 ng and ~1 µg of mutant transcripts were used for the transfection of a flask in (A) and (B), respectively. (C) Altered tetrapeptide CTGK. Fifty to 150 ng of transcript per flask. Days post-transfection are indicated. * the same flask is shown at days 3 and 6. The results presented in panels A–C were obtained in three separate experiments, and some variability of the plaque sizes in the control (TGK-containing) samples could be due to different sensitivities of the cell cultures.

Figure 5. Plaque phenotypes (day 4 post transfection) of viruses in which both the apical loop of domain d of oriL and the TGK motif of 3C were modified.

Figure 6. Efficiency of replication of viral genomes with modifications in only the TGK motif of 3CD (A–C) or in both oriL’s tetraloop and TGK (D). Monolayers of Vero cells were transfected with the RNAs modified as indicated and the accumulation of viral genomes was determined by quantitative PCR. Each point reflects the results obtained in triplicated samples.

Figure 7. Electrophoretic mobility shift assay of the affinity between altered 3CD proteins and wild-type oriL. (A) Illustration of an experiment. The presence of 3CD in the complexes with the mobility indicated has been demonstrated previously by Western blotting [20]. (B) The optical densities of the oriL/3CD complex bands (relative to that of the TGK-containing controls) observed in two experiments (the interaction of oriL with the RTGM-containing 3C was investigated only once).

Figure 8. The space of nucleotide (A) and amino acid (B) sequences corresponding to codons 154–156 of the poliovirus protein 3C. The nucleotides and amino acids in the wild-type poliovirus are given in black. The set of viability-compatible nucleotide substitutions caused by single point mutation and substitutions requiring an additional mutation are given in blue and orange, respectively. The viability-compatible amino acids are in green.

Figure 9. Some features of the crystal structure of 3C. Panel (A) (based on PDB ID 1L1N) demonstrates the proximity of the RNA-binding motif TGK to some other relevant amino acids. Oxygen, nitrogen, and carbon atoms are colored in red, blue, and cyan, respectively, and β-barrels and α-helices in yellow and blue/magenta, respectively. The distances between α carbons of His89, Gly155, Tyr6, β carbon of Cys153, ε carbon of Lys156, C4 of Tyr6, and C5 of His89 are indicated in Å. The electrostatic potentials of the solvent accessible surfaces of proteins 3C of poliovirus (based on PDB ID 4DCD) and rhinovirus (based on PDB ID 1CQQ), shown on panels (B) and (C), respectively, were calculated by using a PyMOL APBS Electrostatics Plugin. The potential range from negative −3 mV (blue) to positive +3 mV (red) is shown at the bottom of panel (C). Relevant residues (see the Discussion section) are highlighted.