Figure 3.

EIAV Rev interacts with equine SAMHD1. (A) GST affinity-isolation analysis of EfSAMHD1 and Rev. Flag-SAMHD1 was co-transfected with VR-GST or Rev-GST. Whole-cells lysates were prepared, and GST affinity isolations were performed with glutathione magbeads and analyzed using western blotting. (B) Co-IP analysis of EfSAMHD1 and Rev. HEK293T cells were transfected with indicated plasmids. Whole-cell lysates were prepared at 48 hpt, and immunoprecipitations were performed with anti-Flag beads and analyzed using western blotting. (C) EIAV Rev relocalizes EfSAMHD1 from the nucleus to cytoplasmic compartments. Flag-EfSAMHD1 and Rev-HA were expressed individually or together in HeLa cells. Cells were fixed, permeabilized and incubated with primary and secondary antibodies at 36 hpt, EfSAMHD1 and Rev expression were observed using confocal microscopy. Scale bar: 5 μm. (D) Schematic of the BiFC fusion proteins. (E) Detection of EfSAMHD1 and Rev interaction using BiFC assay. VN-Flag-EfSAMHD1 and Rev-VC were expressed individually or together in HeLa cells and EfSAMHD1 and Rev fusion proteins were stained with rabbit anti-Flag or anti-HA polyclonal antibodies followed by Alexa Fluor 647-conjuated goat anti-rabbit antibodies. BiFC green fluorescent signals together with the expression of EfSAMHD1 or Rev expression were visualized using confocal microscopy. Scale bar: 5 μm. (F) HIV-1 Rev does not produce BiFC green fluorescent signals with EfSAMHD1. Rev_HIV-VC-HA was used as a negative control. Scale bar: 20 μm