Figure 3
In vitro and in vivo assays to validate VSR activity.
(a) Western blotting to show changes in GFP levels upon transfection with nsP2 and nsP3. Sf21 sensor cell line was transfected with VSRs and western blotting was done using anti-GFP antibody. GADPH was used as housekeeping control. (b and c) CHIKV nsP2 and nsP3 show RNAi suppressor activity in in vivo system. Transgenic Nicotiana leaves with GFPshRNA stably integrated were infiltrated with VSR expressing Agrobacterium cultures and checked for GFP reversion under UV transilluminator. FHVB2 was used as positive control and mutated FHVB2 was the negative control. FHVB2M shows necrosis marks due to infiltration, but no GFP reversion was seen. (d) Northern blotting to show changes in GFP mRNA and small RNA levels upon VSR infiltration in Nicotiana leaves. RNA isolated from infiltrated leaves was used to detect GFP mRNA levels using GFPshRNA oligonucleotide end labelled with [γ32P] ATP. 18 S was used as housekeeping control. GFP small RNA population was detected by northern blotting using 700 bp DIG labelled GFP probe. 28SrRNA was used as house keeping control. (e)Electrophoretic mobility shift assay (EMSA) using labeled GFPshRNA probe and VSR transfected Sf21 cell lysate. GFPshRNA oligonucleotide was end-labelled with [γ32P] ATP and mixed with different concentrations of VSR transfected Sf21 cell lysate. Lane 1: Free shRNA probe; lane 2, 3, 4, 5: Different concentrations of nsP3 (30 μg, 20 μg) and nsP2 (30 μg, 20 μg) transfected Sf21 lysate respectively; lane 6&7: nsP3 and nsP2 transfected Sf21 cell lysate with 100 fold unlabelled GFPshRNA probe; lane 8, 9 & 10: binding of untransfected Sf21 cells to GFPshRNA in the absence and presence (1 μg & 2 μg) of uncompetitive inhibitor. Salmon sperm DNA (2 μg) was used as non specific inhibitor in all binding reactions.
