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.

Virus-like particles (VLPs) were produced as described [26] . Briefly, 293T cells were transfected using Lipofectamine 2000 (Invitrogen) with pCAGGS-BlaM1 (encoding a beta-lactamase reporter protein fused to the influenza matrix protein-1), pCAGGS-HA (encoding HA derived from either A/NewYork/ 1/1918 or IAV-WSN) and pCAGGS-NA (encoding IAV neuraminidase [NA] derived from either A/BrevigMission/1/18 or IAV-WSN) and maintained in OptiMEM. Supernatants were harvested 72 h after transfection and centrifuged to remove debris. VLPs were used for inoculation of cells without further concentration. VLPs were incubated for 30 min at 37uC with trypsin/TPCK for activation of HA. MDCK or HeLa cells grown to near confluency in 24-well plates were inoculated with 250 ul of VLPs after pre-treatment of the cells with inhibitors as indicated. Infection was synchronized by centrifugation at 1500 rpm for 90 min at 4uC and was performed by further incubation at 37uC for 2 h in the absence or presence of 10% FCS and inhibitors as indicated. Detection of beta-lactamase activity was performed as described [25] by loading cells with CCF2-AM substrate (InVitrogen) and subsequent analysis by flow cytometry on a LSRII flow cytometer (Becton Dickinson). Typically 10,000 events were collected and analyzed using FlowJo 8.5.2 software.