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.

Cells were seeded in 96-well plates at a density of 10,000 cells/ well and transfected the next day with 10 ng pHH-Gluc using Lipofectamine 2000 (InVitrogen) according to the manufacturer's protocol. After 24 hrs the transfected cells were treated with inhibitors and infected as indicated. At 16 hr p.i. samples from the supernatant were assayed for luciferase activity using the Renilla Luciferase Assay system (Promega) according to the manufacturer's instructions, and the relative light units (RLU) were determined with a Berthold Centro LB 960 plate luminometer. WR-LUC and VSV-FL were used to inoculate HeLa cells (10,000 cells/well) at an MOI of 2, in complete Dulbecco's Modified Eagle's Medium (DMEM) (Lonza, Biowittaker). After 7 hr the luciferase activity was detected using the SteadyGlo assay kit (Promega). The addition of 10% (v/v) FCS did not change infection levels for both viruses.