Fig. 7.

siRNA downregulation of Rabs of the recycling endosome block ANDV egress. Vero cells were infected with ANDV and at 3 dpi were transfected with the following siRNAs: siGlo (100 nM, RISC-binding deficient siRNA), murine PKR (100 nM, unrelated siRNA), Rab8a (100 nM), Rab8a and b (50 nM each), Rab11a (100 nM), Rab11a and b (50 nM each), and quadruple knockdown Rab8a and b, and Rab11a and b (50 nM each). (A) The cells were lysed at 48 hpt and the amount of Rab8, Rab11, and β-actin were determined by western blot analysis. (B) Intracellular ANDV N levels at 48 hpt were determined by Western blot analysis. Protein levels were normalized to GAPDH. (C) Supernatants were harvested at the time of transfection (0 h) and 48 hpt. Virus titer was determined by immune TCID₅₀ analysis. The fold decrease in virus titer between 0 h and 48 h was determined. Data presented is the mean of four independent experiments with duplicate wells analyzed for each condition.