Figure 4.

EIAV Rev promotes the lysosomal degradation of equine SAMHD1. (A) Lysosome pathway inhibitors block Rev-induced EfSAMHD1 degradation. HEK293T cells were transfected with indicated plasmids. At 20 hpt, cells were treated with MG132 (10 μM), 3-MA (5 mM), CQ (150 μM), Baf A1 (200 nM), or wortmannin (200 nM). After incubation for 16 h, the cells were analyzed using western blotting with indicated antibodies. The densities of SAMHD1 bands were analyzed to calculate the values relative to that of ACTB. Results were normalized to respective control cells. (B) EfSAMHD1 is targeted to lysosomes for degradation. VR1012-mcherry or VR1012-LAMP2-mcherry was expressed in HeLa cells with the VN-Flag-EfSAMHD1 and Rev-VC pair. Colocalization of these fluorescent signals was visualized by confocal microscopy. Scale bar: 5 μm. (C and D) Analysis of the effect of protein degradation inhibitors on the SAMHD1-Rev BiFC punctate number. VN-Flag-EfSAMHD1 and Rev-VC were co-expressed in HeLa cells. At 20 hpt, cells were further treated with indicated inhibitors. After Incubation for 16 h, the cells were observed using confocal microscopy (C). Scale bar: 20 μm. The number of SAMHD1-Rev BiFC puncta was statistically analyzed (D)