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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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CoV-induced macropinocytosis is dependent on EGFR activation. (A) Gefitinib was added to cells for 12 h, and toxicity was assessed with CellTiter-Glo. (B, C) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or gefitinib at 1.5 hpi. Cells were fixed at 8 hpi. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. The percentage of cells with internalized nanoparticles (B) and the number of nuclei per syncytium (C) were determined. (D, E) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or EIPA at 1.5 hpi. Cells were fixed at 8 hpi, stained, and imaged. The percentage of infected cells involved in syncytia (D) and the number of nuclei per syncytium (E) were determined. Data are means ± standard deviations of triplicates. n = ≥30 fields per replicate. Statistical 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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fig7: CoV-induced macropinocytosis is dependent on EGFR activation. (A) Gefitinib was added to cells for 12 h, and toxicity was assessed with CellTiter-Glo. (B, C) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or gefitinib at 1.5 hpi. Cells were fixed at 8 hpi. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. The percentage of cells with internalized nanoparticles (B) and the number of nuclei per syncytium (C) were determined. (D, E) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or EIPA at 1.5 hpi. Cells were fixed at 8 hpi, stained, and imaged. The percentage of infected cells involved in syncytia (D) and the number of nuclei per syncytium (E) were determined. Data are means ± standard deviations of triplicates. n = ≥30 fields per replicate. Statistical significance was assessed by one-way ANOVA with Dunnett’s post hoc test. ***, P < 0.0001; **, P < 0.01; *, P < 0.05.

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.


Coronaviruses induce entry-independent, continuous macropinocytosis.

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

CoV-induced macropinocytosis is dependent on EGFR activation. (A) Gefitinib was added to cells for 12 h, and toxicity was assessed with CellTiter-Glo. (B, C) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or gefitinib at 1.5 hpi. Cells were fixed at 8 hpi. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. The percentage of cells with internalized nanoparticles (B) and the number of nuclei per syncytium (C) were determined. (D, E) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or EIPA at 1.5 hpi. Cells were fixed at 8 hpi, stained, and imaged. The percentage of infected cells involved in syncytia (D) and the number of nuclei per syncytium (E) were determined. Data are means ± standard deviations of triplicates. n = ≥30 fields per replicate. Statistical 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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Related In: Results  -  Collection

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fig7: CoV-induced macropinocytosis is dependent on EGFR activation. (A) Gefitinib was added to cells for 12 h, and toxicity was assessed with CellTiter-Glo. (B, C) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or gefitinib at 1.5 hpi. Cells were fixed at 8 hpi. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. The percentage of cells with internalized nanoparticles (B) and the number of nuclei per syncytium (C) were determined. (D, E) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or EIPA at 1.5 hpi. Cells were fixed at 8 hpi, stained, and imaged. The percentage of infected cells involved in syncytia (D) and the number of nuclei per syncytium (E) were determined. Data are means ± standard deviations of triplicates. n = ≥30 fields per replicate. Statistical significance was assessed by one-way ANOVA with Dunnett’s post hoc test. ***, P < 0.0001; **, P < 0.01; *, P < 0.05.
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.

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