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.

BafA1 specifically inhibits IAV during the entry phase as demonstrated in Fig. 1C . The continuous presence of 10 nM BafA1 (added to the cells 1 hr prior to infection) for 16 hrs completely prevents infection. In contrast the addition of BafA1 at 1 hr or 2 hrs post infection resulted in high levels of luciferase activity (again measured at 16 hrs p.i.) that were 63% or 90% respectively of the control to which no BafA1 was added, indicating that entry was essentially completed within 2 hrs. The last bar of Fig. 1C shows that the inhibition by BafA1 is reversible as withdrawal of the inhibitor after 2 hrs resulted in high levels of infection. The specific effect of BafA1 on IAV entry was confirmed by confocal microscopy demonstrating that BafA1, as expected, traps IAV particles in a peri-nuclear location, presumably in nonacidified endosomes (Fig. 1D) . BafA1 was subsequently exploited to establish a specific IAV entry assay (hereafter further referred to as the Gluc-entry assay). HeLa cells transfected with pHH-Gluc were inoculated with IAV at a range of MOIs and incubated for 2 hrs after which the entry medium was replaced by complete growth medium containing 10% FCS and 10 nM BafA1 to prevent any further entry of virus. Entry was indirectly quantified by determination of luciferase activity after further incubation for 14 hrs demonstrating a quantitative correlation between infection dose and luciferase activity across a wide range of MOIs (Fig. 1E) . The indirect Gluc-entry assay was next tested for its capacity to examine the effects of inhibitors on IAV entry. Dynasore or BafA1 (Fig. 1F) were included in the medium (DMEM containing 10%