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: Signaling through the EGFR is essential for induction of macropinocytosis in several systems (31–33). To test whether EGFR activation is required for CoV-induced macropinocytosis, we utilized gefitinib, which specifically inhibits EGFR autophosphorylation and prevents EGFR activation (34). DBT cells were mock infected or infected with MHV A59, gefitinib was added after viral entry at 1.5 hpi, and cells were analyzed for nanoparticle uptake and syncytium size (Fig. 7A to C). Addition of gefitinib to MHV-infected cells significantly decreased the percentage of syncytia with internalized nanoparticles (Fig. 7B). We also observed that gefitinib significantly decreased the number of nuclei in a syncytium, with only half as many nuclei as in a DMSO-treated syncytium (Fig. 7C). We then evaluated the effect of EIPA on syncytium size and discovered that treatment with EIPA decreased both the percentage of infected cells involved in syncytia (Fig. 7D) and the size of the syncytia (Fig. 7E). These data suggest that CoVs utilize EGFR activation to induce macropinocytosis and to initiate cell-to-cell spreading.