The Most Abnormal Ceramidase Saga

Consistent with the mutagenic effect of ribavirin, we found a significant reduction in plaque size for WT, Max, and SD (p?Ceramidase of a virus population within sequence space determines its tolerance of mutation. We next examined whether fitness differences among the spectrum of mutants generated by each poliovirus synonym result in fitness differences among the three populations. In direct competition assays (Figure?3A), the Max population was somewhat fitter than the WT (wrel 1.74?�� 0.17, n?= 5 replicates), and SD was markedly less fit (wrel 0.18?�� 0.02, n?= 5 replicates). We also observed these fitness differences at a 10-fold higher multiplicity of infection (moi ?10), where frequent coinfection results in complementation of defective genomes and proteins. Here, Max exhibited a fitness closer to the WT reference (1.054?�� 0.004, n?= 5 replicates), Anticancer Compound Library datasheet while SD remained considerably less fit (0.712?�� 0.019, n?= 5 replicates). These results are consistent with the idea that the less robust SD population is less fit because of Selleckchem AZD8055 the impact of mutation on the variants generated during each replication cycle. An alternative explanation for the observed fitness differences, however, would be a direct effect of specific synonymous mutations on viral replication or translation. We therefore measured the kinetics of genome replication in a single replication cycle, using a sensitive and precise quantitative polymerase chain reaction assay (Figure?3B). We found no statistically significant difference in the exponential growth rate between WT and either SD or Max (p?= 0.1837 and p?= 0.5178, respectively). Despite their 10-fold difference in relative fitness, Max and SD had nearly identical growth curves, with only small differences evident at the plateau phase of replication (see Figure?S1 online). In contrast, we were able to measure a replication defect for a poliovirus mutant with a much more modest fitness difference. Under identical assay conditions, this guanidine-resistant poliovirus (2CN179G) exhibits a fitness of 0.56 and a significant lag in replication relative to WT (Figure?S1, Pincus et?al., 1986; Lauring and Andino, 2011). To exclude a subtle translation defect in SD, we directly assayed viral capsid translation in infected and metabolically labeled cells (Figure?3C).