Coronaviruses Lacking Exoribonuclease Activity Are Susceptible to Lethal Mutagenesis: Evidence for Proofreading and Potential Therapeutics
(A) 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) or 5-FU (C) 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). (D) 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 t test (*P<0.05, **P<0.01, ***P<0.0001).