Fig. 2

Confirmation of both intracellular and extracellular interactions between RDRP and host proteins. a Yeast two-hybrid testing of RDRP-host protein interactions. Yeast strain Y187 was co-transformed with pGADT7-RDRP and pGBKT7-prey and plated on SD/-Trp/-Leu/-His/-ADE/-x-α-gal plates. Positive interactions were indicated by the presence of blue colonies. The co-transformation of pGBKT7-53 and pGADT7-T was used as a positive control, whereas the co-transformation of pGBKT7-Lam and pGADT7-T was used as a negative control. b GST pull-down assay confirming extracellular interactions between RDRP and OTU. The binding of His-RDRP to GST-OTU immobilised on glutathione beads was determined by Western blot analysis. The total protein eluted from the beads was probed with an anti-His antibody as indicated. c The intracellular interaction between RDRP and host OTU proteins as determined by coimmunoprecipitation. DH10Bac bacteria were transfected with pFast-HTA-RDRP and pFast-dual-OTU to construct the corresponding recombinant bacmids. The recombinant bacmids were then extracted and transfected into Sf9 cells. The recombinant baculoviruses were incubated with transfected cells at 27 °C and harvested every 7 days. Third-generation viruses were employed to infect Sf9 cells for protein expression. After co-transfection of OTU and RDRP recombinant baculoviruses for 48 h, cell lysates were analysed by Western blotting with anti-His and anti-GST antibodies. Immunoprecipitation was performed with an anti-His antibody (d) and an anti-GST antibody (e) and detected by Western blotting with anti-His and anti-GST antibodies. Untransfected cells were used as controls