Fig 3. Effects of TOSV NSs N-terminal domain on E3 ubiquitin ligase activity.

(A) The involvement of NSs Cystein₂₇ in the ubiquitination of RIG-I was evaluated by immunofluorescence on cells co-transfected with RIG-I and NSs plasmids. Cells were stained with anti-FLAG antibody and RIG-I positive cells were counted on different fields. Mean values ± SD of more than three independent experiments were plotted (left panel). Results were validated by immunoblotting (right panel) using total cell lysates of co-transfected cells and semi-quantitative analysis was done by densitometry (supplement data 4). (B) Lenti-X 293T cells were transfected with IFN-β reporter plasmid (p125-FFLuc) along with RIG-I and pSV40-RenLuc plasmids in addition with the wild type-NSs, or C₂₇G-NSs mutant or empty plasmids. After stimulation with poly(I:C), luciferase activity was analyzed. For each sample, luciferase was normalized to the RenLuc reporter activity. Data are representative of three independent experiments and are expressed as mean ± SD of normalized luciferase activity.