Molecular and Functional Bases of Selection against a Mutation Bias in an RNA Virus

Abstract

The selective pressures acting on viruses that replicate under enhanced mutation rates are largely unknown. Here, we describe resistance of foot-and-mouth disease virus to the mutagen 5-fluorouracil (FU) through a single polymerase substitution that prevents an excess of A to G and U to C transitions evoked by FU on the wild-type foot-and-mouth disease virus, while maintaining the same level of mutant spectrum complexity. The polymerase substitution inflicts upon the virus a fitness loss during replication in absence of FU but confers a fitness gain in presence of FU. The compensation of mutational bias was documented by in vitro nucleotide incorporation assays, and it was associated with structural modifications at the N-terminal region and motif B of the viral polymerase. Predictions of the effect of mutations that increase the frequency of G and C in the viral genome and encoded polymerase suggest multiple points in the virus life cycle where the mutational bias in favor of G and C may be detrimental. Application of predictive algorithms suggests adverse effects of the FU-directed mutational bias on protein stability. The results reinforce modulation of nucleotide incorporation as a lethal mutagenesis-escape mechanism (that permits eluding virus extinction despite replication in the presence of a mutagenic agent) and suggest that mutational bias can be a target of selection during virus replication.

Keywords: 5-fluorouracil; antiviral resistance; fitness; foot-and-mouth disease virus; lethal mutagenesis; protein folding stability.

Fig. 1.—

Passage history, acquisition of mutations in the polymerase (3D)-coding region, and fitness of FMDV-3D(V173I) relative to FMDV-wt in the presence of different FU concentrations. (A) FMDV-RP (its origin is described in Materials and Methods) was subjected to 15 passages in the absence or presence of 5-fluorouracil (FU) (50 µg/ml or 200 µg/ml added to culture medium) to yield populations RP-15, RP(FU50)-15 and RP(FU200)-15, respectively. (B) Amino acid substitutions in 3D of populations depicted in A, deduced from the corresponding consensus sequence of the 3Dcoding region; +, presence of mutation; −, absence of mutation; +/ −, presence of mutation in ∼50% of the RNA population, according to the corresponding nucleotide peak in the sequence. Numbering of genomic and amino acid residues is according to Escarmís et al. (1999). (C) To determine the relative fitness of FMDV-3D(V173I) and FMDV-wt, BHK-21 cells were infected with a mixture of FMDV-3D(V173I) and FMDV-wt; the RNA ratio of the two viruses ranged between 1:1 and 0.1:1, at a total initial MOI of 0.1 PFU/cell. Passages were performed in the presence of the concentration of FU (µg/ml) indicated below each bar. RNA of the two competing viruses was measured in triplicate. Fitness values were calculated as described in Materials and Methods; the fitness determination plots are given in supplementary figure S2, Supplementary Material online. The error bars indicate the error of the fit of each individual fitness value. Statistical significances are computed through the t-test, to evaluate if the slope of the ratio of RNA of the two competing viruses versus the passage number (nine points) is different from zero; significances are represented as (*P < 0.05; **P < 0.01). The statistical significance of the difference between pairs of fitness values is indicated on the lines linking two bars (*P < 0.05; **P < 0.005; Welch’s two-tailed test); P values above 0.05 were obtained in the comparison of fitness values obtained with 400 µM FU versus 200 µM FU, and 200 µM FU versus 100 µM FU. Procedures are further detailed in Materials and Methods.

Fig. 2.—

Presteady-state kinetics of nucleotide incorporation into sym/sub-UA and sym/sub-FuG by 3Dwt and 3D(V173I). (A) Sequence of 5′-end-labeled, annealed sym/sub-UA. Arrows indicate the template residue at which nucleotide incorporation is measured. (B) 3D (0.5 µM active sites) was preincubated at 37 °C with sym/sub-UA (0.5 µM duplex) and ATP (10 µM) for 900 s to allow the formation of 3D-RNA product complex, with AMP incorporated at the first position. The 3D-RNA product was then mixed with the indicated concentration of UTP or FUTP using a rapid chemical quench-flow apparatus, and reactions were quenched by the addition of EDTA (0.3 M). Independent time points at 0, 0.01, 0.05, 0.1, 0.25, 0.5, 1, and 2 s were taken for each nucleotide concentration tested. Time courses at fixed nucleotide concentrations were fit to an exponential curve to obtain the observed rate constant for nucleotide incorporation at the second position, k_obs. The observed rate constants were then plotted as a function of nucleotide concentration, and the data were fit to a hyperbola to obtain k_pol and K_d,app. Note that the scale at ordinate is different in the two panels. (C) Sequence of 5′-end-labeled, annealed sym/sub-FuG (Fu means FU). Arrows indicate the template residue at which nucleotide incorporation is measured (FU is written F). (D) Incorporation of AMP or GMP; no nucleotide was preincubated with 3D and RNA. Procedures are those described in (B). For the assays with GTP, the concentration of 3D used was 1 µM active sites, and no rapid chemical quench-flow apparatus was needed. Duplicate samples at 0, 10, 30, 60, 120, 300 and 900 s time points were taken for each nucleotide concentration tested. Note that the scale in ordenate is different in the two panels. Procedures are further detailed in Materials and Methods.

Fig. 3.—

Structure of FMDV-3D(V173I). (A) The left and right panels show two views of 3D(V173I) protein rotated by 90°. The polymerase is depicted in blue ribbons with substituted amino acid I173 shown in sticks in red. The triphosphate moiety of the bound ATP and the motif F contacting side chains R168 and K172 are also shown as sticks and explicitly labeled. (B) Stereoview of σA-weighted |Fo| − |Fc| electron density map (contoured at 3σ) around the mutated residue I173. The substituted residues and surrounding amino acids were omitted from the phasing model. The model is placed inside in ball and stick representation and colored in atom type code. (C) Stereoview of the structural changes around the mutated I173 residue. 3D(V173I) polymerase is shown in white (with I173 depicted in red) and the superimposed 3Dwt in cyan (TP refers to the ATP triphosphate moiety). (D) Superimposition of the N-terminal region (residues E11–K20) of the FMDV-wt polymerase (cyan) and the FMDV-3D(V173I) polymerase (white). Information on data collection and refinement statistics is given in supplementary table S3, Supplementary Material online. The PDB accession codes for the 3Ds represented here are 1WNE for 3Dwt and 5DTN for 3D(V173I).

Fig. 4.—

Predicted effect of transition mutations and non-synonymous mutations in the FMDV genome and 3D-coding region. (A) Fraction of non-synonymous mutations for each transition in the 3D-coding region and the entire FMDV genome; statistical significances are given in the text. (B) Mean change in folding free energy (ΔΔG; negative values correspond to stabilizing substitutions) resulting from all possible non-synonymous transitions in the 3D-coding region, as predicted by PoPMuSiC and DeltaGREM. Both programs agree qualitatively on the comparison between different transitions, although DeltaGREM tends to predict more mutations as being stabilizing (ΔΔG < 0). Procedures are detailed in Materials and Methods.