IFITM3 Clusters on Virus Containing Endosomes and Lysosomes Early in the Influenza A Infection of Human Airway Epithelial Cells

Kummer, Susann; Avinoam, Ori; Kräusslich, Hans-Georg

Influenza A virus A/Hong Kong/1/1968 (HK/1/68), A/Puerto Rico/8/1934 (PR/8/34) and A/Regensburg/ D6/2009 (R/D6/09) were recovered in a HEK 293T-MDCK-coculture using the eight plasmid reverse genetics systems, as previously described [62] . In live-cell experiments, Cell Mask™ Orange plasma membrane stain (catalogue no. C10045, Life Technologies, sigma-aldrich, Steinheim, Germany) was directly added to the cell supernatant prior to the virus purification, following the manufacturer's guidelines. The virus stocks were titrated, making use of a standard plaque assay with minor changes, as previously described [63] . Briefly, serial 10-fold dilutions of each virus stock were prepared in DMEM supplemented with 0.3% bovine serum albumin (BSA, sigma-aldrich, Steinheim, Germany), 20 mM HEPES (sigma-aldrich, Steinheim, Germany), penicillin (200 U/mL)/streptomycin sulphate (100 µg/mL) (Capricorn scientific GmbH, Ebsdorfergrund, Germany) and TPCK-trypsin (2 µg/mL) (sigma-aldrich, Steinheim, Germany). MDCK cell monolayers were incubated with 200 µL of each dilution for 1 h at 37 • C in a 6 well plate format. Then, the cells were overlaid with 2 mL of 2.4% Avicel (FMC) in aqua dest. and 2 × DMEM (Merck, Darmstadt, Germany) in a 1:1 ratio. After 5 days, the overlay was removed, the cells were fixed with 1 mL/well of 70% ethanol for 30 min at room temperature and stained with 1 mL/well of 0.3% crystal violet (sigma-aldrich, Steinheim, Germany) in 20% methanol for 10 min at room temperature.