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.

Macropinocytosis has been implicated in the entry of several viruses [12, 14] . However, differences in susceptibility to inhibitors suggest that distinct forms of macropinocytosis might be used by different viruses [34, 35] . By screening specific inhibitors in the Gluc-entry assay using DYNA-IND entry conditions we evaluated the possible involvement of a few signaling cascades that have been implicated in the induction of macropinocytosis. Serum-inducible macropinocytosis has been shown to be activated via a myriad of signaling cascades initiated by growth factors binding to transmembrane tyrosine kinase receptors [14, 17, 36, 37] , consistent with the results shown in Fig. 5 . A prominent downstream effect of these signaling cascades is the activation of p21 associated kinase 1 (PAK1) which in turn can activate a number of different pathways leading to actin network rearrangements that can ultimately lead to the induction of macropinocytosis [38] . Fig. 9A -B shows that 20 mM IPA3, an inhibitor of PAK1 [39] , specifically inhibits Background fluorescence from Fdx binding to the outside of cells was determined by performing the same experiment at 4uC (at which no endocytosis takes place) and was subtracted from the mean fluorescence intensity obtained at 37uC to determine the amount of fluorescent FITC-dextran that was internalized at 37uC. Data were plotted relative to FITCdextran uptake in PBS in absence of IAV. doi:10.1371/journal.ppat.1001329.g008 DYNA-IND entry of IAV. Activation of PAK1 in response to growth factor stimulation often involves upstream signal transduction by members of the Rho sub-family of small GTPases like CDC42 and/or Rac1 [34, 40, 41] . Alternatively, activated CDC42 and Rac1 can induce actin rearrangements independently of PAK1 [34, [40] [41] [42] [43] by direct interaction with WASP or WAVE family proteins, respectively [44, 45] . However, inhibitors of CDC42 (Pirl1 [46] ), Rac1 (NSC23766 [47] ) or N-WASP (wiskostatin [48] ) did not display inhibitory effects on DYNA-IND or DYNA-DEP entry of IAV (Fig. 9C-D) . Instead, Pirl1 and wiskostatin induced a significant, concentration dependent increase of entry. This stimulatory effect was not observed for the control vaccinia virus strain WR, which enters cells via a Rac1dependent, macropinocytotic pathway [43] (Fig. 9E) , indicating that this effect is specific for IAV. The results suggest a requirement for PAK1 in DYNA-IND entry of IAV that does not require activation by either CDC42 or Rac1. Growth factor inducible activation of the tyrosine kinase src has also been linked to the induction of macropinocytosis [49] [50] [51] ; consistent with this observation the src inhibitor PP2 [52] specifically inhibited DYNA-IND entry of IAV (Fig. 9A-B) . Remarkably, 17-AAgeldanamycin, a specific inhibitor of the chaperone protein HSP90 [53] , also caused specific inhibition of DYNA-IND entry (Fig. 9A-B) . HSP90 affects the folding and activity of many proteins but the recent demonstration of direct activation of the catalytic activity of src by HSP90 [54] provides another indication of the involvement of src in DYNA-IND endocytosis of IAV.