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Coronaviruses Lacking Exoribonuclease Activity Are Susceptible to Lethal Mutagenesis: Evidence for Proofreading and Potential Therapeutics

Figure 2

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

(A) 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) 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. (C) 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 [33]. 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 t test (*P<0.05, **P<0.01, ***P<0.0001).

Figure 2