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.

In response to specific extra-cellular signals (e.g. serum induction), changes in the actomyosin network occur that give rise to membrane protrusions required for macropinosome formation [13] . Compounds inhibiting actin polymerization (cytochalasin B and D), depolymerization (jasplakinolide) or sequestering soluble actin (latrunculin A) all specifically inhibited DYNA-IND IAV entry. In addition, the requirement for myosinII activity was established by a specific inhibitor (Blebbistatin) of myosin II ATPase activity and by the expression of a dominant negative mutant of myosinIIA heavy chain. Also, the regulation of myosinII activity by phosphorylation of myosin light chain through the action of MLCK is suggested by the inhibitory effect of MLCK inhibitors ML-7 and ML-9 as well as by the similar effect of an expressed MLCK dominant negative mutant. Recently, a function for the actin cytoskeleton in IAV entry was reported to be required for the entry into polarized epithelial cells but not for entry into non-polarized cells [64] . When using the low-serum conditions used in that paper (2% FCS), we only observed DYNA-DEP entry that was not affected by actin dynamics inhibitors. Perhaps, the polarized cells permit DYNA-IND entry at lower serum concentrations.