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: To test for the role of spike protein and spike protein fusogenic activity in MHV-induced macropinocytosis, we mock infected or infected cells with MHV A59 or with recombinant MHV A59 encoding the MHV-2 spike protein (MHV 2S) (29) and tested nanoparticle internalization. MHV 2S-infected cells internalized significantly fewer nanoparticles than MHV A59-infected cells (Fig. 5A). To test the specific requirement for spike protein cleavage, we infected cells with an MHV A59 C12 mutant containing an amino acid mutation (H716D) that abolishes the furin cleavage site (30). During infection with MHV C12, there was no significant difference in nanoparticle internalization from that in mock-infected cells (Fig. 5A). We next used a furin inhibitor, peptidyl chloromethyl ketone (dec-RVKR-cmk) (27) to block furin cleavage of the MHV A59 spike protein. Treatment with dec-RVKR-cmk decreased both nanoparticle uptake and syncytium formation in a concentration-dependent manner (Fig. 5B to D). To test whether spike protein cleavage alone is sufficient to induce macropinocytosis, we treated cells infected with MHV 2S with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin in order to generate fusogenic spike protein on the cell surface and assessed nanoparticle uptake. Cells infected with MHV 2S and treated with trypsin recovered the capacity for nanoparticle internalization (Fig. 5E). Together, these results demonstrate that expression of fusion-competent spike protein at the plasma membrane is necessary for MHV macropinocytosis induction.