FIG 4 .

MHV-ExoN(-) evolved WT-like genomic RNA accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using TRIzol at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).