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.

HeLa cells were grown in 24-well plates on glass coverslips (50,000 cells/well). Prior to FITC-dextran uptake cells were serum-starved for 2 hr in PBS. FITC-dextran (MW70,000, Sigma-Aldrich) was incubated with HeLa cells (final concentration of 0.5 mg/ml) in 500 ml PBS or in PBS containing 10% FCS in the absence or presence of IAV (strain WSN; MOI 10; concentrated and purified by centrifugation through a 15 to 30% sucrose gradient with a 50% sucrose cushion at the bottom) at 37uC. After 15 min cells were washed 4 times with PBS at 4uC, fixed with 3.7% PFA in PBS and subsequently permeabilized with 0.1% Triton-X-100 in PBS. Slides were stained for examination by confocal microscopy as described above. For quantification of FITC-dextran uptake 1.5610 5 HeLa cells were infected with IAV-WSN (MOI 10) in suspension in a volume of 1 ml in the presence of FITC-Dextran (1 mg/ml). Infections were performed for 15 min in PBS (containing 2% BSA to reduce unspecific binding of FITC-Dextran) or in PBS containing 10% FCS at 37uC or at 4uC (control for binding of FITC-Dextran to cells in the absence of endocytosis). Mock-infected samples were analysed in parallel. Infection was terminated by addition of 3 ml ice-cold PBS followed by three washes with cold PBS and fixation with 3.7% PFA. 20,000 cells were analyzed by FACS and results were represented as the mean fluorescence which was plotted relative to the uptake in the mock-infection in PBS (after subtraction of background fluorescence obtained at 4uC).