Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

de Vries, Erik; Tscherne, Donna M.; Wienholts, Marleen J.; Cobos-Jiménez, Viviana; Scholte, Florine; García-Sastre, Adolfo; Rottier, Peter J. M.; de Haan, Cornelis A. M.

Two siRNA duplexes targeting different sites within the coding sequences of dynamin 2 were obtained from Ambion Inc (15581 (Dynamin 2 siRNA 1) and 146559 (dynamin 2 siRNA2)). A scrambled siRNA (Ambion Inc.) was taken along as a control for non-specific effects of the transfection procedure and was used for normalization. One day after seeding in 96-well plates (6,000 cells/well), the HeLa cells were transfected with a final concentration of 10 nM siRNA using oligofectamine (Invitrogen). 48 h after transfection, the cells were inoculated with the WSN-Ren pseudovirus (MOI 0.5) in PBS or in PBS containing 10% FCS. After 2 h of infection the entry medium was replaced by complete growth medium containing 10 nM BafA1 to prevent further entry. At 16 h post infection intracellular Renilla luciferase expression was determined as described above. Each siRNA experiment was performed in triplicate. Cell viability was not affected as determined by performing a Wst-1 cell-viability assay (Roche). Functional knockdown of dynamin 2 mRNA levels was performed by quantitative RT-PCR. using a TaqMan Gene Expression Assay for DNM2 (Hs00191900_m1, Ambion) and using 18S RNA (Hs03928985_g1, Ambion) as a control for normalization. The comparative Ct-method was used for quantification of the results [77] . Reduction of dynamin 2 protein levels was determined by western blotting using polyclonal goat-anti-dynamin 2 C18 (Santa-Cruz SC-6400). A monoclonal against alpha-tubulin (DM1A, Sigma T9026) was used to detect tubulin for normalization. Results were quantified by Densitometric scanning of the dynamin 2 and tubulin signals displayed in Fig. 4H .