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.

The changes in actin network dynamics that can lead to the formation of macropinosomes can be triggered by a number of signaling cascades. Actin dynamics are induced by the activation of growth factor receptor tyrosine kinases by their respective Figure 10 . A model for IAV entry by macropinocytosis. The model summarizes the inhibitory (red boxes) or stimulatory (blue boxes) effects of compounds on dynamin-independent IAV entry. The effect of over-expression of dominant-negative mutants is indicated by red-lined boxes. The pathway requires the presence of serum factors in the entry medium and results in the enhanced uptake of dextran and its co-localization with IAV in large vesicles (green boxes). We hypothesize that the interaction of serum factors and/or IAV with receptor tyrosine kinases (RTKs) is the primary signal for the induction of macropinocytosis. A number of RTKs have been shown to be involved in this process in different cell lines. Remarkably, a recently published genome-wide siRNA screen of IAV infection identified the FGF receptor as a host factor required for influenza virus replication [22] . Activation of Rho family GTPases CDC42 and/or Rac1 has been shown to be essential for signal transduction leading to macropinocytosis in many cases [13, 14] but inhibitors are without effect or are stimulatory in the case of IAV entry. Downstream effectors of Rho family GTPases include scaffold proteins like N-WASP and WAVE and protein kinases like PAK1. Macropinocytic entry of IAV however seems to require a Rho family GTPaseindependent PAK1 activation mechanism. In addition, src family kinases, which can be directly activated by RTKs, play a role. PAK1 and src have previously been linked to the activation of macropinocytosis via their effect on changes in actomyosin dynamics, a process which is crucial to any form of macropinosome formation [13, 14] . Apart from N-WASP-or WAVE-containing macromolecular assemblies other actin binding proteins can induce such changes (e.g. cortactin, which can be activated by src [81] ) and thereby induce the formation of one of the different plasma membrane protrusions that can result in the formation of macropinosomes. In addition to an effect on the formation of plasma membrane protrusions and subsequent macropinosome formation, inhibitors can also affect downstream trafficking and maturation of macropinosomes which might be actindependent, but this is not depicted in the scheme. doi:10.1371/journal.ppat.1001329.g010 growth factor ligands that are normally present in serum [12] [13] [14] 17, 36, 37] The signal transduction cascades that link activation of growth factor receptor tyrosine kinases to actin remodeling and macropinocytosis are only beginning to be revealed. The specific inhibition of DYNA-IND entry of IAV by IPA3, an inhibitor of PAK1, provides proof for the involvement of these cascades. PAK1 is a key serine/threonine kinase regulating actin network dynamics but its crucial function in several pathways of endocytosis as well as numerous other cellular processes does not make it a very specific marker [65] . Even so, macropinocytosis has consistently been demonstrated to require PAK1 activation, both in the induction of the process and/or in further downstream trafficking events of macropinosomes [13, 14] .