Figure 7

αE mutations completely inhibit WNV replication. (a) HEK293T cells were electroporated with wild-type (WT) or αE mutant (αE Mut) WNV RLuc2A replicon RNA. Cells were lysed at indicated time points post-transfection, and the total luciferase activity in 10 μg total protein from cell lysate was measured in relative light units (RLUs) by luciferase assay. Each sample was read in triplicate, and figure is representative of two experimental replications. Statistical significance is represented as follows: *, P< 0.0005. (b) Capped WNV RNA corresponding to the first 190 nucleotides of the WNV genome was used to examine both N-7 (lanes 2–4) and 2′-O (lanes 6–8) methylation. For N-7 methylation, capped WNV RNA was incubated in the presence of SAM with either mock (1), Vaccinia virus capping enzyme (VCE) (2), wild-type WNV methyltransferase (3), or WNV αE mutant methyltransferase (4). For 2′-O methylation, m7GpppA-WNV RNA was incubated in the presence of SAM with mock (5), Vaccinia 2′-O methyltransferase (VMT) (6), wild-type WNV methyltransferase (7) or WNV αE mutant methyltransferase (8). The reactions were run out by TLC on PEI cellulose and ability of αE mutant WNV methyltransferase to generate both m7GpppA and m7GpppAm was compared to wild-type WNV.