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Coronaviruses induce entry-independent, continuous macropinocytosis.

Freeman MC, Peek CT, Becker MM, Smith EC, Denison MR - MBio (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: mark.denison@vanderbilt.edu.

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Related in: MedlinePlus

The presence of fusogenic spike protein at the plasma membrane is required to induce macropinocytosis. (A) Cells were mock infected or infected with MHV A59, 2S, or C12 at an MOI of 1 PFU/cell. Nanoparticles were added at 5 hpi for 3 h, and the cells were washed, fixed, stained, and imaged. (B to D) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM with DMSO or dec-RVKR-cmk (dRc) for 8 h. The cells were exposed to dec-RVKR-cmk for 12 h, and toxicity was assessed with CellTiter-Glo (B). Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Percentages of syncytial cells (C) and cells with internalized nanoparticles (D) were measured. (E) Cells were mock infected or infected with MHV A59 or 2S at an MOI of 1 PFU/cell. At 5 hpi, cells were treated with TPCK trypsin for 5 min and washed and then nanoparticles were added for 3 h. Cells were washed, fixed, stained, and imaged. Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. n = ≥30 cells per replicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test; ***, P < 0.0001; **, P < 0.01; *, P < 0.05.
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fig5: The presence of fusogenic spike protein at the plasma membrane is required to induce macropinocytosis. (A) Cells were mock infected or infected with MHV A59, 2S, or C12 at an MOI of 1 PFU/cell. Nanoparticles were added at 5 hpi for 3 h, and the cells were washed, fixed, stained, and imaged. (B to D) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM with DMSO or dec-RVKR-cmk (dRc) for 8 h. The cells were exposed to dec-RVKR-cmk for 12 h, and toxicity was assessed with CellTiter-Glo (B). Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Percentages of syncytial cells (C) and cells with internalized nanoparticles (D) were measured. (E) Cells were mock infected or infected with MHV A59 or 2S at an MOI of 1 PFU/cell. At 5 hpi, cells were treated with TPCK trypsin for 5 min and washed and then nanoparticles were added for 3 h. Cells were washed, fixed, stained, and imaged. Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. n = ≥30 cells per replicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test; ***, P < 0.0001; **, P < 0.01; *, P < 0.05.

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.


Coronaviruses induce entry-independent, continuous macropinocytosis.

Freeman MC, Peek CT, Becker MM, Smith EC, Denison MR - MBio (2014)

The presence of fusogenic spike protein at the plasma membrane is required to induce macropinocytosis. (A) Cells were mock infected or infected with MHV A59, 2S, or C12 at an MOI of 1 PFU/cell. Nanoparticles were added at 5 hpi for 3 h, and the cells were washed, fixed, stained, and imaged. (B to D) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM with DMSO or dec-RVKR-cmk (dRc) for 8 h. The cells were exposed to dec-RVKR-cmk for 12 h, and toxicity was assessed with CellTiter-Glo (B). Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Percentages of syncytial cells (C) and cells with internalized nanoparticles (D) were measured. (E) Cells were mock infected or infected with MHV A59 or 2S at an MOI of 1 PFU/cell. At 5 hpi, cells were treated with TPCK trypsin for 5 min and washed and then nanoparticles were added for 3 h. Cells were washed, fixed, stained, and imaged. Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. n = ≥30 cells per replicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test; ***, P < 0.0001; **, P < 0.01; *, P < 0.05.
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fig5: The presence of fusogenic spike protein at the plasma membrane is required to induce macropinocytosis. (A) Cells were mock infected or infected with MHV A59, 2S, or C12 at an MOI of 1 PFU/cell. Nanoparticles were added at 5 hpi for 3 h, and the cells were washed, fixed, stained, and imaged. (B to D) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM with DMSO or dec-RVKR-cmk (dRc) for 8 h. The cells were exposed to dec-RVKR-cmk for 12 h, and toxicity was assessed with CellTiter-Glo (B). Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Percentages of syncytial cells (C) and cells with internalized nanoparticles (D) were measured. (E) Cells were mock infected or infected with MHV A59 or 2S at an MOI of 1 PFU/cell. At 5 hpi, cells were treated with TPCK trypsin for 5 min and washed and then nanoparticles were added for 3 h. Cells were washed, fixed, stained, and imaged. Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. n = ≥30 cells per replicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test; ***, P < 0.0001; **, P < 0.01; *, P < 0.05.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: mark.denison@vanderbilt.edu.

Show MeSH
Related in: MedlinePlus