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.

Growth factor dependent activation of PAK1 has most often been demonstrated to depend on upstream activation of small GTPases Rac1 or cdc42 [34, 40, 41] . Different strains of vaccinia virus were recently shown to induce their uptake by macropinocytosis via activation of either Rac1 or cdc42 [34] . Activation of Rac1 has been linked to the induction of macropinocytosis via actin network-mediated formation of lamellipodia and/or circular ruffles whereas cdc42 has most often been implied in the formation of filopodia [44] . An inhibitory effect of the Rac1 inhibitor NSC23766 or the cdc42 inhibitor pirl1 on IAV entry, however, could not be demonstrated. Remarkably, cdc42 inhibitor pirl1 enhanced IAV entry and a similar effect was observed by wiskostatin, an inhibitor of N-WASP which functions directly downstream of cdc42 as a scaffolding complex required for the activation of actin polymerization leading to filopodia formation. Similarly, the macropinocytosis-like entry pathway taken by Coxsackie B virus was also shown to require PAK1 activity that was independent of Rac1 activation [35] . Direct examination of the magnitude and timing of the activation of PAK1 will be required to obtain more insight in the involvement of this complex pathway. The induction of macropinocytosis by a PAK1dependent mechanism has been associated with ruffling at the cell membrane [12, 14, 15, 37] . The identification of sub-membranous regions with increased actin staining by phalloidin has been interpreted as evidence for ruffling. This was not unambiguously identified by confocal microscopy in the experiments presented in Fig. 8 and Fig. S2 and needs to be investigated in depth by life cell imaging techniques.