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.

Our evidence for a DYNA-DEP and a serum inducible DYNA-IND entry route is based on the use of pharmacological (dynasore, a highly specific inhibitor of dynamin) as well as genetic (siRNA directed against dynamin 2) tools, ruling out the possibility that the inhibitory effect of dynasore was due for instance to absorption of the inhibitor by serum components. Whereas dynasore resulted in near 100% inhibition of DYNA-DEP entry, only 65% inhibition was observed upon siRNA induced silencing of dynamin 2 indicating that the residual levels of dynamin 2 that remain after 48 hrs of silencing still support a low level of DYNA-DEP entry (Fig. 4H) . Reversible inhibitors like dynasore [60] offer a major advantage for characterization of IAV entry pathways. They can be applied for a limited period thus preventing the secondary adaptive effects of cells that may occur in response to long-term down regulation of a gene product by genetic methods like siRNA interference. Both entry routes were consistently identified by a viral entry assay quantified by virus induced expression of a luciferase reporter as well as by a VLP entry assay allowing direct analysis of the membrane fusion mediated entry step. The consistent performance of an HA with a strict preference for binding to a2-3 linked sialic acids (from IAV-WSN; our unpublished data) and an HA also binding to a2-6 linked sialic acids (from 1918 IAV [61] ) in the VLP entry assay indicates that both pathways can be utilized by HAs of different specificity and may therefore be relevant to avian as well as human IAV infections. Consistently, serum-inducible DYNA-IND entry was observed both in avian DF1 cells and in a human lung epithelial carcinoma cell line A549 (Fig. 3) . The DYNA-DEP and DYNA-IND IAV entry pathways were found by our quantitative assays to be fully redundant. In the presence of serum, the combination of dynasore (inhibiting DYNA-DEP entry) and EIPA (inhibiting DYNA-IND entry) completely abolished entry whereas either drug alone had no effect. EIPA, an inhibitor of plasma membrane Na+/H+ exchangers, has been shown to invariably inhibit macropinocytosis [14, [55] [56] [57] [58] . As other routes of endocytosis are generally not affected, EIPA is considered as a hallmark inhibitor of macropinocytosis [14] , although results obtained with EIPA should be considered with care as long as a mechanistic explanation for its effect on macropinocytosis is not yet fully clear [62] . Occasionally, a moderate two-to three-fold inhibition by dynasore alone was observed (result not shown) indicating that the capacity of the serum-inducible entry pathway is somewhat variable, possibly depending on slight variations in serum quality and factors like cell distribution in the wells that have been reported to influence viral infection [63] . A redundancy in the utilization of CME as well as a clathrin-independent route for entry of IAV has been visualized previously by quantitative live cell imaging [4] . Both routes were operative simultaneously in the same sample and the specific down-regulation of CME did not affect the total number of entry events.