FIG 4.

APOL1 depletes intracellular Vif by lysosomal degradation and stimulating its secretion in microvesicles. (A) 293T cells were transfected with Vif-expressing pNL-A1 vector alone or in combination with APOL1 expression vector. Five hours after transfection, the cells were treated with E64d (10 μg/ml) and pepstatin A (10 μg/ml). After 24 h, whole-cell lysates and pelleted microvesicles were separated by 10% SDS-PAGE and analyzed by Western blotting for the expression of Vif, APOL1, and microvesicle markers Alix and CD81. Secretion of microvesicles was inhibited by incubation of transfected cells with the calcium chelator BAPTA-AM (10 μM) for 16 h. (B) Depletion of Rab7A with siRNA inhibited lysosomal degradation of Vif and potentiated APOL1-mediated release of Vif in microvesicles. 293T cells were transfected twice with Rab7A siRNA, followed by transfection with pNL-A1 alone or with APOL1 vector. After 24 h, whole-cell lysates and pelleted microvesicles were separated by 10% SDS-PAGE and analyzed by Western blotting for the expression of Vif, APOL1, and Rab7 and microvesicle markers Alix and Tsg101. Co, control.