5-FU-mediated U:C and A:G transitions are distributed across the CoV genome at low frequency.

<p>(<b>A</b>) and (<b>B</b>) The genomic distribution of low frequency statistically significant U:C and A:G variants within the SARS-ExoN+ population following treatment with 0 or 400 µM 5-FU. (<b>C</b>) and (<b>D</b>) Same as in A and B except for the SARS-ExoN− population. For all panels, SARS-ExoN+ viruses are shown in blue, and SARS-ExoN− viruses are shown in green. U:C transitions are denoted by a diamond, whereas A:G transitions are plotted as circles.</p

The increased sensitivity of MHV-ExoN− viruses to 5-FU is consistent with mutagenesis.

<p>(<b>A</b>) DBT cells in 96-well plates were incubated with DMEM alone, or DMEM containing 20% ethanol (EtOH), 4% DMSO, or the indicated concentration of 5-FU for 12 h. Cell viability was determined using CellTiter-Glo (Promega) according to manufacturer's instructions. All values were normalized to the untreated (DMEM) control. Mean values ± S.E.M. are shown, n = 2. (<b>B</b>) MHV-ExoN+ (filled circle) and MHV-ExoN− (open circle) virus sensitivity to 5-FU during single- (solid lines; MOI = 1 PFU/cell) and multi-cycle (dotted lines; MOI = 0.01 PFU/cell) replication. MHV-ExoN+ viruses are shown in blue and MHV-ExoN− viruses are shown in green. The change in virus titer was calculated by dividing virus titers following treatment by the untreated controls. Mean values ± S.E.M. are shown, n = 4. (<b>C</b>) The change in titer (filled bars) and genomic RNA levels (hatched bars) of MHV-ExoN+ (blue) and MHV-ExoN− (green) viruses following treatment with 5-FU is shown. DBT cells were infected with MHV-ExoN+ or MHV-ExoN− in the presence or absence of 5-FU, and virus titer was determined by plaque assay. Genomic RNA levels were determined using two-step real-time qRT-PCR and primers optimized to amplify a ∼120 nt region of ORF1a <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003565#ppat.1003565-Donaldson1" target="_blank">[33]</a>. The change in genomic RNA levels (2^−ΔΔCt) is shown relative to endogenous GAPDH expression and was normalized to RNA levels from untreated samples. Mean values ± S.E.M. are shown, n = 6. For all parts, statistical significance was determined using an unpaired, two-tailed Student's <i>t</i> test (*P<0.05, **P<0.01, ***P<0.0001).</p

The antiviral activity of RBV against ExoN− viruses is not primarily due to mutagenesis.

<p>(<b>A</b>) DBT cells in 96-well plates were incubated with DMEM alone, or DMEM containing 20% ethanol (EtOH), 4% DMSO, or the indicated concentration of RBV for 12 h. Cell viability was determined using CellTiter-Glo (Promega) according to manufacturer's instructions. All values were normalized to the untreated (DMEM) control. No significant differences were found when RBV-treated values were compared to DMEM samples containing DMSO (+DMSO) using an unpaired, two-tailed Student's <i>t</i> test. Mean values ± S.E.M. are shown, n = 2. (<b>B</b>) MHV-ExoN+ (filled circle) and MHV-ExoN− (open circle) virus sensitivity to RBV during single- (solid lines; MOI = 1 PFU/cell) and multi-cycle (dotted lines; MOI = 0.01 PFU/cell) replication. MHV-ExoN+ viruses are shown in blue and MHV-ExoN− viruses are shown in green. The change in virus titer was calculated by dividing virus titers following treatment by the untreated controls. Mean values ± S.E.M. are shown, n = 4. (<b>C</b>) The change in titer (filled bars) and genomic RNA levels (hatched bars) of MHV-ExoN+ (blue) and MHV-ExoN− (green) viruses following treatment with RBV is shown. DBT cells were infected with MHV-ExoN+ or MHV-ExoN− in the presence or absence of RBV, and virus titer was determined by plaque assay. Genomic RNA levels were determined using two-step real-time qRT-PCR and primers optimized to amplify a ∼120 nt region of ORF1a <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003565#ppat.1003565-Donaldson1" target="_blank">[33]</a>. The change in genomic RNA levels (2^−ΔΔCt) is shown relative to endogenous GAPDH expression and was normalized to RNA levels from untreated samples. Mean values ± S.E.M. are shown, n = 6. (<b>D</b>) MHV-ExoN+ (filled circle) and MHV-ExoN− (open circle) virus sensitivity to mycophenolic acid (MPA) during single- (solid lines; MOI = 1 PFU/cell) and multi-cycle (dotted lines; MOI = 0.01 PFU/cell) replication. Mean values ± S.E.M. are shown, n = 2–4. RBV- or MPA-treated MHV-ExoN+ (<b>E</b>) and MHV-ExoN− (<b>F</b>) viruses with or without the addition of 100 µM guanosine (GUA) during single-cycle replication (MOI = 1 PFU/cell). Mean values ± S.E.M. are shown, n = 2. For all parts, statistical significance was determined using an unpaired, two-tailed Student's <i>t</i> test (*P<0.05, **P<0.01, ***P<0.0001).</p

Incorporation of FUMP results in increased U:C and A:G transitions.

<p>All possible base changes are shown for SARS-ExoN+ and SARS-ExoN− viruses in panels (<b>A</b>) and (<b>B</b>), respectively. Transitions (A↔G and U↔C) are shaded in grey, and 5-FU specific transitions (U:C and A:G) are marked with an asterisk. Transversions (A↔T, A↔C, C↔G, G↔T) are shown in white boxes. All values represent the number of unique statistically significant minority variants following 5-FU treatment. (<b>C</b>) The percent of all unique statistically significant minority variants represented by transversions (filled dark grey bars), C:U and G:A transitions (filled light grey bars), and the 5-FU specific transitions A:G (hatched bars) and U:C (checkered bars) are shown following 0 or 400 µM 5-FU treatment. SARS-ExoN+ viruses are shown in blue, and SARS-ExoN− viruses are shown in green.</p

SARS-ExoN− viruses have increased sensitivity to 5-FU.

<p>(<b>A</b>) Vero cells in 96-well plates were incubated with DMEM alone, or DMEM containing 20% ethanol (EtOH), 4% DMSO, or the indicated concentration of RBV or 5-FU for 24 h. Cell viability was determined using CellTiter-Glo (Promega) according to manufacturer's instructions. All values were normalized to the untreated (DMEM) control. Mean values ± S.E.M. are shown, n = 3. The change in SARS-ExoN+ (filled blue circles) and SARS-ExoN− (empty green circles) titers following treatment with RBV (<b>B</b>) or 5-FU (<b>C</b>) during single-cycle replication. Vero cells were infected with either virus at an MOI of 0.1 PFU/cell, and virus supernatant was harvest 24 h post-infection following replication in the presence or absence of RBV or 5-FU. Virus titer was determined by plaque assay on Vero cells. Mean values ± S.E.M. are shown, n = 2 (RBV) and n = 4 (5-FU). (<b>D</b>) Comparison of unique statistically significant (P<0.05) minority variants present between untreated and 5-FU treated samples for both SARS-ExoN+ and ExoN− populations. SARS-ExoN+ viruses are shown in blue, and SARS-ExoN− viruses are shown in green. For panels A–C statistical significance was determined using an unpaired, two-tailed Student's <i>t</i> test (*P<0.05, **P<0.01, ***P<0.0001).</p

Mutagenizing position 483 variants allows isolation of mutator variants.

<p>(A) HeLa cells, treated with 200 µM or 400 µM ribavirin, or left untreated, were infected at an MOI of 0.1. The percentage of infectious progeny virus surviving treatment at both concentrations of ribavirin relative to the untreated control is shown (mean values ± SEM, n = 3, *P at least<0.05, two-way ANOVA with Bonferroni posttest). ND, not determined. (B) Average mutation frequencies of WT CHIKV and variants with significantly altered fidelity. Mutation frequency is shown as the mean number of mutations per 10,000 nucleotides sequenced by molecular cloning. Variants 483A, G and W made significantly more errors than WT virus (*P<0.05, **P<0.01, ***P<0.001, χ² test). (C) Average diversity of confirmed fidelity variants at each position across the genome. The root-mean-square-deviation (RMSD) is shown (mean values ± SEM, ***P<0.001, Mann-Whitney <i>u</i> test). (D) Neutralization assay showing enhanced escape of mutators due to greater population diversity (mean values ± SEM, *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA with Bonferroni posttest).</p

Mutagenizing SINV position C482 generates mutator strains that exhibit replication defects in mosquito cells.

<p>(A) Alignment of five alphaviruses at the conserved cysteine residue at position 482/483 (SINV/CHIKV) in CHIKV, SINV, Semliki forest virus (SFV), O'nyong nyong virus (ONNV), and Venezuelan equine encephalitis virus (VEEV). (B) Ribavirin sensitivity of potential SINV mutators. HeLa cells, treated with 200 µM or 400 µM ribavirin, or left untreated, were infected at an MOI of 0.1. The percentage of infectious progeny virus surviving treatment at both concentrations of ribavirin relative to the untreated control is shown (mean values ± SEM, n = 3, *P at least<0.05, two-way ANOVA with Bonferroni posttest). (C) Average mutation frequencies determined by molecular clone sequencing of WT SINV and variants 482A and G. Approximately 75 clones were sequenced per variant (*P<0.05, χ2 test). (D–G) Viral titers determined by TCID₅₀ (D and F) and RNA genomes determined by qRT-PCR (E and G) of SINV mutators in (D and E) BHK-21 cells and (F and G) <i>Ae. albopictus</i> C6/36 cells. Mutator variants exhibit significant defects in C6/36 but these defects are reduced in BHK-21 cells (mean values ± SEM, n = 3, **P<0.01, ***P<0.001, two-way ANOVA with Bonferroni posttest).</p

Mutators are under strong pressure to revert in mosquito models.

<p>(A–C) <i>Ae. aegypti</i> or (D–F) <i>Ae. albopictus</i> were infected with WT and mutator strains of CHIKV, and mosquitoes were collected at 1, 3, 7 and 14 days post-infection. <i>Ae. aegypti</i> mosquitoes exhibited no significant differences in RNA genomes in (A) bodies, (B) legs and wings, or (C) saliva at 7 days post-infection. Seven days post-infection <i>Ae. albopictus</i> mosquitoes exhibited no differences in RNA genomes in (D) bodies with the exception of a significant but slight decrease for 483A. There were no significant differences observed in the (E) legs and wings or (F) saliva (median values ± IQR, n = 10, *P<0.001, two-way ANOVA with Bonferroni posttest).</p

Mutator variants 483A, G, and W are attenuated in mice.

<p>(A) Seven days post-infection, mutators have significantly fewer RNA genomes present in the muscle, blood, brain, and liver of newborn mice compared to WT (median values ± IQR are shown, n = 4, *P<0.05, **P<0.01, two-way ANOVA with Bonferroni posttest). (B) Average mutation frequencies of WT CHIKV and mutators 483A, G and W from the muscle of a mouse representing the median titer of each group. All mutators retain an elevated mutation frequency, with the most attenuated variant (C483W) making significantly more errors than WT virus (**P<0.01, χ² test).</p

Evolutionary rates of RABV genes in the dog-related group.

<p>(<b>A</b>) Rates of nucleotide substitution per site, per year were estimated for each RABV gene: nucleoprotein (N), phosphoprotein (P), matrix (M), glycoprotein (G) and polymerase (L), for the concatenated non-coding regions (NC) and for the five concatenated RABV genes (5 genes). Both the mean and the 95% highest posterior density (HPD) values on the rate are shown. (<b>B</b>) Substitution rates in the N and G genes of the dog-related group RABV, a sub-set of RABV circulating in mongooses (MG) in Africa-3 clade and in the Caribbean, in ferret-badgers (FB) in Asia, and in dogs in Asia and Africa. Note the different y-axes (rates) in both cases.</p