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.

Several dynamin-independent endocytic pathways have been described [8, 19] . Of these, macropinocytosis has been demonstrated to be stimulated by growth factors present in serum and to depend on actin dynamics [12] [13] [14] . Yet, studies on macropinocytosis are hampered by a lack of specific inhibitors, cargo, membrane markers and characteristic morphology. Amiloride and the more potent derivative EIPA are inhibitors of epithelial sodium channels (ENaC) as well as of several other Na+/H+ antiporters. EIPA has often been used as a hallmark inhibitor that specifically inhibits endocytosis via the macropinocytic pathway [14] . Whereas DYNA-DEP entry of IAV was not inhibited by EIPA (Fig. 7A) , DYNA-IND entry was fully blocked EIPA (Fig. 7B) . The existence of redundant entry pathways in the presence of 10% FCS is clearly demonstrated by the marginal inhibition by either EIPA or dynasore whereas the combination of EIPA and dynasore resulted in strong inhibition both in the Gluc-entry assay (Fig. 7C ) and in the direct VLP entry assay ( Fig. 7D and E) . Supplementary Fig. S1 shows that other cell lines, including the human lung epithelial cell line A549, display similar IAV inhibition patterns for EIPA and dynasore. Consistently, virus production displayed a similar inhibitor sensitivity profile (Fig.7 F and G) as virus entry indicating that the entry pathways we characterized lead to a productive infection. Clearly, VLPs and viral particles follow similar redundant entry pathways, distinguishable in a DYNA-DEP and a DYNA-IND pathway, the latter being sensitive to EIPA and dependent on actomyosin function.