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.

MDCK, A549, DF-1 and HeLa cells were maintained in complete Dulbecco's Modified Eagle's Medium (DMEM) (Lonza, Biowittaker) containing 10% (v/v) fetal calf serum (FCS; Bodinco B.V.), 100 U/ml Penicillin, and 100 mg/ml Streptomycin. Chinese Hamster-E36 cells were maintained at 37uC in a-Minimal Essential Medium (Gibco) supplemented with 10% (v/v) FCS, 100 U/ml Penicillin, and 100 mg/ml Streptomycin. Cells were passaged twice weekly. Influenza A/WSN/33 (H1N1) (IAV-WSN) was grown in MDCK cells. Briefly, ,70% confluent MDCK cells were infected with IAV-WSN at a MOI of 0.02. Supernatant was harvested after 48 hr of incubation at 37uC and cell debris was removed by centrifigutation (10 min at 2000 rpm). Virus was stored at 280uC and virus titers were determined by measuring the TCID 50 on HeLa cells. The IAV-WSN luciferase pseudovirus (WSN-Ren) system has previously been described [27] . Briefly, WSN-Ren pseudovirus harbors a HA segment in which the HA coding region is replaced by Renilla luciferase. The pseudovirus is produced in a MDCK cell line that stably expresses the HA of IAV-WSN. WR-LUC, a firefly luciferase encoding vaccinia virus (strain WR) was previously described [75] . VSV-FL, a firefly luciferase encoding VSV virus was also previously described [76] .