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.

As factors present in serum are known for their potential to induce specific endocytic pathways, we further explored the conditions required for the novel DYNA-IND IAV entry pathway (using the Gluc-entry assay) by inoculating cells in PBS in the presence of increasing concentrations of fetal calf serum (FCS). Whereas dynasore completely inhibited entry in PBS, inclusion of 5% and 10% FCS resulted in increasing levels of dynasore resistant entry ( Fig. 2A) , suggesting the existence of a serum-inducible DYNA-IND IAV entry pathway. This effect was not caused by inactivation of dynasore during the experiment as vesicular stomatitis virus (VSV), which enters cells by CME [24, 25] , was still sensitive to 80 mM dynasore in the presence of 10% FCS (Fig. 2B) . In agreement herewith, the uptake of transferin, known to occur via CME, was inhibited by dynasore regardless of the HeLa cells were grown on glass cover slips and infected with IAV (strain WSN; MOI of 10) and fixated after 30 min, 3 hrs or 6 hrs (column 1, 2 or 3 respectively). Infection was performed in 0.2% DMSO (upper row panels) or in the presence of 10 nM BafA1 (lower row panels). The nucleus was visualized by DNA staining with TOPRO-3 (red). IAV infection was visualized by staining with monoclonal antiserum directed against NP (green). In the absence of inhibitor, IAV localized to the nucleus after 3 hrs, while new virus particles spread to the cytoplasm after 6 hrs. BafA1 (lower row panels) caused accumulation of incoming virus particles at a peri-nuclear location. (E) Quantitative determination of IAV entry by a single-cycle Gluc-entry assay. HeLa cells (10,000 cells/well in DMEM supplemented with 10% FCS) were transfected with pHH-Gluc 24 hrs prior to infection with a serial dilution of infectious IAV particles (plotted on the x-axis). Two hours after infection 10 nM BafA1 was added to block any further entry. Cells were incubated for a further 14 hrs to allow expression of luciferase activity (y-axis; Relative Light Units, RLU). (F) Effect of Dynasore and BafA1 on IAV entry in the Gluc-entry assay. Dynasore (DY, dark grey bars; 20, 40 or 80 mM) or BafA1 (light grey bars; 1.25, 2.5 or 5 nM) were present from 1 hr prior to infection (strain WSN; MOI 0.5) to 2 hrs p.i. after which the inhibitor-containing medium was replaced with medium containing 10 nM BafA1 to block any further entry. Cells were incubated for a further 14 hrs to allow the quantitative expression of luciferase activity (y-axes; RLU relative to the control infection without inhibitor). Whereas BafA1 displayed dose-dependent inhibition of IAV entry, dynasore did not significantly inhibit IAV entry. presence of FCS (Fig. S2, panel A) . As expected, both DYNA-DEP entry in PBS and DYNA-IND entry in the presence of 10% FCS and 80 mM dynasore required sialic acid receptors for efficient entry as pre-treatment of HeLa cells with neuraminidases almost completely abolished entry via either pathway (Fig. 2C ). The kinetics of the DYNA-DEP and DYNA-IND entry pathways were compared by performing a time-course experiment in which IAV entry was terminated by the addition of 10 nM BafA1 at different time points (Fig. 2D) . In comparison to entry via the DYNA-DEP pathway (the only pathway available in PBS) entry in the presence of FCS (when presumably both the DYNA-DEP and DYNA-IND entry pathways are available) showed similar kinetics. In contrast, entry via the DYNA-IND pathway (which is the only pathway that is active in the p