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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

MHV-induced macropinocytosis is dependent on the classical macropinocytosis pathway. (A, B) Cells were reverse transfected with siRNA for 72 h, and protein knockdown was confirmed by immunoblotting (A) and standardized to GAPDH (B). Scrambled siRNA (sc)- and siRNA (si)-treated samples are from the same gel for each protein. RhoA and Pak1 are from discontinuous lanes separated by dashed lines. Data are represented as the means ± the standard errors of the means in triplicate assays. (C) Cells were reverse transfected for 68 h and infected with MHV for 8 h. Nanoparticles were added during the final 3 h, and cells were washed, fixed, stained, and imaged. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate, n = ≥30 fields per replicate. (D) The 12-h toxicity of EIPA was assessed with CellTiter-Glo. (E) Cells were mock infected or infected with MHV at an MOI of 1 PFU/cell for 8 h with no drug, DMSO, or 40 µM EIPA. Nanoparticles were added during the final 3 h of infection. Cells were washed, fixed, stained, and imaged, and the percentage of cells with internalized nanoparticles was calculated. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.
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fig3: MHV-induced macropinocytosis is dependent on the classical macropinocytosis pathway. (A, B) Cells were reverse transfected with siRNA for 72 h, and protein knockdown was confirmed by immunoblotting (A) and standardized to GAPDH (B). Scrambled siRNA (sc)- and siRNA (si)-treated samples are from the same gel for each protein. RhoA and Pak1 are from discontinuous lanes separated by dashed lines. Data are represented as the means ± the standard errors of the means in triplicate assays. (C) Cells were reverse transfected for 68 h and infected with MHV for 8 h. Nanoparticles were added during the final 3 h, and cells were washed, fixed, stained, and imaged. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate, n = ≥30 fields per replicate. (D) The 12-h toxicity of EIPA was assessed with CellTiter-Glo. (E) Cells were mock infected or infected with MHV at an MOI of 1 PFU/cell for 8 h with no drug, DMSO, or 40 µM EIPA. Nanoparticles were added during the final 3 h of infection. Cells were washed, fixed, stained, and imaged, and the percentage of cells with internalized nanoparticles was calculated. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.

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.


Coronaviruses induce entry-independent, continuous macropinocytosis.

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

MHV-induced macropinocytosis is dependent on the classical macropinocytosis pathway. (A, B) Cells were reverse transfected with siRNA for 72 h, and protein knockdown was confirmed by immunoblotting (A) and standardized to GAPDH (B). Scrambled siRNA (sc)- and siRNA (si)-treated samples are from the same gel for each protein. RhoA and Pak1 are from discontinuous lanes separated by dashed lines. Data are represented as the means ± the standard errors of the means in triplicate assays. (C) Cells were reverse transfected for 68 h and infected with MHV for 8 h. Nanoparticles were added during the final 3 h, and cells were washed, fixed, stained, and imaged. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate, n = ≥30 fields per replicate. (D) The 12-h toxicity of EIPA was assessed with CellTiter-Glo. (E) Cells were mock infected or infected with MHV at an MOI of 1 PFU/cell for 8 h with no drug, DMSO, or 40 µM EIPA. Nanoparticles were added during the final 3 h of infection. Cells were washed, fixed, stained, and imaged, and the percentage of cells with internalized nanoparticles was calculated. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.
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fig3: MHV-induced macropinocytosis is dependent on the classical macropinocytosis pathway. (A, B) Cells were reverse transfected with siRNA for 72 h, and protein knockdown was confirmed by immunoblotting (A) and standardized to GAPDH (B). Scrambled siRNA (sc)- and siRNA (si)-treated samples are from the same gel for each protein. RhoA and Pak1 are from discontinuous lanes separated by dashed lines. Data are represented as the means ± the standard errors of the means in triplicate assays. (C) Cells were reverse transfected for 68 h and infected with MHV for 8 h. Nanoparticles were added during the final 3 h, and cells were washed, fixed, stained, and imaged. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate, n = ≥30 fields per replicate. (D) The 12-h toxicity of EIPA was assessed with CellTiter-Glo. (E) Cells were mock infected or infected with MHV at an MOI of 1 PFU/cell for 8 h with no drug, DMSO, or 40 µM EIPA. Nanoparticles were added during the final 3 h of infection. Cells were washed, fixed, stained, and imaged, and the percentage of cells with internalized nanoparticles was calculated. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.
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.

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