Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme

Stewart, Meredith E.; Roy, Polly

As NYR mutant virus replication showed the most drastic effect on replication in comparison to the WT virus, we examined the viral protein synthesis at different time points. At 48 h p.i., both structural (e.g., VP3) and non-structural proteins (e.g., NS1) were detected in cell infected with the mutant virus although at levels lower than that of WT (Fig. 7C) , consistent with the low virus titres. In order to quantify the difference in protein expression between the NYR mutant and WT viruses, a more sensitive Renilla luciferase assay was undertaken [2] . Cells were transfected with an S10-RLuc, an ssRNA that expresses Renilla luciferase under the regulation of BTV NS1, followed by infection with NYR mutant or WT BTV. Renilla luciferase production was quantified 12 h p.i (Fig. 7D) . In contrast to NYR mutant virus and control, Renilla luciferase activity was $4-fold higher in the WT BTV infected cells (Fig. 7D) . These results confirmed that the mutations affecting the growth of the viruses also perturb the viral protein production during early infection.