Coronaviruses induce entry-independent, continuous macropinocytosis.
Bottom Line: MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells.These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading.In this work, we show that CoVs induce a macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased virus titers and cell fusion.
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Mentions: We next determined whether MHV-induced macropinocytosis requires known mediators of cellular macropinocytosis. We selected Rac1, Cdc42, and Pak1 from the classical macropinocytosis pathway for small interfering RNA (siRNA) inhibition. Inhibition of RhoA was chosen as a negative control, since it is not associated with macropinocytosis. For each siRNA molecule, a target knockdown rate of ≥80% was confirmed by immunoblotting (Fig. 3A and B). Transfection efficiency was tested with siRNA-AllStars-GFP and found to be >96% (data not shown). Inhibition of Pak1, Cdc42, and Rac1 resulted in significantly decreased nanoparticle internalization following MHV infection, while nanoparticle uptake was unchanged in cells transfected with a scrambled siRNA or with a siRNA targeting RhoA (Fig. 3C). These results demonstrate that MHV-induced macropinocytosis signals through a known cellular macropinocytosis pathway.