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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

Infection with MHV or SARS CoV induces bulk fluid uptake consistent with macropinocytosis. (A, B) DBT cells were infected with MHV A59 at an MOI of 1 PFU/cell for 8 h. DBT-hACE2 cells were infected with SARS CoV at an MOI of 0.1 PFU/cell for 24 h. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Arrows denote nanoparticles (red). DAPI is blue, and nsp8 is green. (B) Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. n = ≥30 fields per replicate. (C) Cells were mock infected or infected with MHV, UV-inactivated MHV (UV), or heat-inactivated MHV (HI) at an MOI = 1 PFU/cell (or the equivalent volume of noninfectious virus) for 8 h. Nanoparticles were added in 2-h increments, as designated, and 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. (D) DBT cells on a glass bottom dish were infected with DiI-labeled MHV (white arrows) at an MOI of 25 PFU/cell, incubated at 4°C for 30 min, and imaged in a 37°C chamber incubator for 1 h. Time is in minutes:seconds. Virus fusion with the cell was observed, but membrane ruffling was absent. An image representative of triplicate experiments is shown. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. **, P < 0.005; ***, P < 0.0001.
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fig2: Infection with MHV or SARS CoV induces bulk fluid uptake consistent with macropinocytosis. (A, B) DBT cells were infected with MHV A59 at an MOI of 1 PFU/cell for 8 h. DBT-hACE2 cells were infected with SARS CoV at an MOI of 0.1 PFU/cell for 24 h. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Arrows denote nanoparticles (red). DAPI is blue, and nsp8 is green. (B) Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. n = ≥30 fields per replicate. (C) Cells were mock infected or infected with MHV, UV-inactivated MHV (UV), or heat-inactivated MHV (HI) at an MOI = 1 PFU/cell (or the equivalent volume of noninfectious virus) for 8 h. Nanoparticles were added in 2-h increments, as designated, and 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. (D) DBT cells on a glass bottom dish were infected with DiI-labeled MHV (white arrows) at an MOI of 25 PFU/cell, incubated at 4°C for 30 min, and imaged in a 37°C chamber incubator for 1 h. Time is in minutes:seconds. Virus fusion with the cell was observed, but membrane ruffling was absent. An image representative of triplicate experiments is shown. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. **, P < 0.005; ***, P < 0.0001.

Mentions: A hallmark of macropinocytosis is bulk fluid uptake from the surrounding environment. To test whether CoV-infected cells were inducing bulk fluid uptake, we used Nile Red neutral polystyrene nanoparticles with a diameter of 800 nm as markers, since their size is excluded from all endocytic pathways except macropinocytosis. MHV-infected DBT cells (Fig. 2A and B) and SARS CoV-infected DBT-hACE2 cells (Fig. 2B) were incubated with nanoparticles during the last 3 h of infection. Cell monolayers infected with either MHV or SARS CoV demonstrated a significantly higher percentage of cells with internalized nanoparticles than mock-infected cells (Fig. 2B). Macropinocytosis has been described as a means of pathogen entry in several different systems. However, since our data suggested that MHV-induced ruffling was occurring much later during infection, we next sought to determine the timing of bulk fluid uptake during MHV infection. Cells were mock infected or infected with MHV and then incubated with nanoparticles for 2-h intervals beginning at infection and fixed immediately afterward (Fig. 2C). Significantly increased nanoparticle uptake was first detected between 4 and 6 hpi and was also prominent at 6 to 8 hpi, indicating that increased fluid-phase uptake initiates at 4 h or later postentry and continues through the remainder of infection. This result was consistent with our visual and quantitative measurements of ruffling and vesicle formation. Because nanoparticle uptake occurred late during infection, we next tested whether replicating MHV is required to induce bulk fluid uptake. Cells were mock infected, infected with MHV, or incubated with an equal concentration of UV-inactivated or heat-inactivated MHV and incubated with nanoparticles for 2-h intervals, beginning at infection (Fig. 2C). UV-inactivated MHV did not result in increased nanoparticle uptake, suggesting that surface receptor interactions were insufficient to induce macropinocytosis. Heat-inactivated MHV also did not cause nanoparticle internalization, suggesting that the membrane-ruffling phenotype observed was not a cellular response to foreign particles. In addition, in order to confirm that macropinocytosis is not utilized for entry of CoVs, we labeled virions with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and added them to DBT cells grown on a glass bottom dish at a high multiplicity of infection (MOI) of 25 PFU/cell (Fig. 2D). Virions were adsorbed for 30 min at 4°C to synchronize the infection before they were transferred to the 37°C chamber incubator surrounding the microscope. Cells were then live imaged for 1 h. While we did visualize viruses entering cells at the plasma membrane, we did not observe any evidence of cellular membrane ruffling at the site of entry or elsewhere on the cell. Thus, virus infection is required for induction of bulk fluid uptake, and MHV-induced macropinocytosis is not associated with virus entry.


Coronaviruses induce entry-independent, continuous macropinocytosis.

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

Infection with MHV or SARS CoV induces bulk fluid uptake consistent with macropinocytosis. (A, B) DBT cells were infected with MHV A59 at an MOI of 1 PFU/cell for 8 h. DBT-hACE2 cells were infected with SARS CoV at an MOI of 0.1 PFU/cell for 24 h. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Arrows denote nanoparticles (red). DAPI is blue, and nsp8 is green. (B) Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. n = ≥30 fields per replicate. (C) Cells were mock infected or infected with MHV, UV-inactivated MHV (UV), or heat-inactivated MHV (HI) at an MOI = 1 PFU/cell (or the equivalent volume of noninfectious virus) for 8 h. Nanoparticles were added in 2-h increments, as designated, and 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. (D) DBT cells on a glass bottom dish were infected with DiI-labeled MHV (white arrows) at an MOI of 25 PFU/cell, incubated at 4°C for 30 min, and imaged in a 37°C chamber incubator for 1 h. Time is in minutes:seconds. Virus fusion with the cell was observed, but membrane ruffling was absent. An image representative of triplicate experiments is shown. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. **, P < 0.005; ***, P < 0.0001.
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fig2: Infection with MHV or SARS CoV induces bulk fluid uptake consistent with macropinocytosis. (A, B) DBT cells were infected with MHV A59 at an MOI of 1 PFU/cell for 8 h. DBT-hACE2 cells were infected with SARS CoV at an MOI of 0.1 PFU/cell for 24 h. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Arrows denote nanoparticles (red). DAPI is blue, and nsp8 is green. (B) Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. n = ≥30 fields per replicate. (C) Cells were mock infected or infected with MHV, UV-inactivated MHV (UV), or heat-inactivated MHV (HI) at an MOI = 1 PFU/cell (or the equivalent volume of noninfectious virus) for 8 h. Nanoparticles were added in 2-h increments, as designated, and 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. (D) DBT cells on a glass bottom dish were infected with DiI-labeled MHV (white arrows) at an MOI of 25 PFU/cell, incubated at 4°C for 30 min, and imaged in a 37°C chamber incubator for 1 h. Time is in minutes:seconds. Virus fusion with the cell was observed, but membrane ruffling was absent. An image representative of triplicate experiments is shown. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. **, P < 0.005; ***, P < 0.0001.
Mentions: A hallmark of macropinocytosis is bulk fluid uptake from the surrounding environment. To test whether CoV-infected cells were inducing bulk fluid uptake, we used Nile Red neutral polystyrene nanoparticles with a diameter of 800 nm as markers, since their size is excluded from all endocytic pathways except macropinocytosis. MHV-infected DBT cells (Fig. 2A and B) and SARS CoV-infected DBT-hACE2 cells (Fig. 2B) were incubated with nanoparticles during the last 3 h of infection. Cell monolayers infected with either MHV or SARS CoV demonstrated a significantly higher percentage of cells with internalized nanoparticles than mock-infected cells (Fig. 2B). Macropinocytosis has been described as a means of pathogen entry in several different systems. However, since our data suggested that MHV-induced ruffling was occurring much later during infection, we next sought to determine the timing of bulk fluid uptake during MHV infection. Cells were mock infected or infected with MHV and then incubated with nanoparticles for 2-h intervals beginning at infection and fixed immediately afterward (Fig. 2C). Significantly increased nanoparticle uptake was first detected between 4 and 6 hpi and was also prominent at 6 to 8 hpi, indicating that increased fluid-phase uptake initiates at 4 h or later postentry and continues through the remainder of infection. This result was consistent with our visual and quantitative measurements of ruffling and vesicle formation. Because nanoparticle uptake occurred late during infection, we next tested whether replicating MHV is required to induce bulk fluid uptake. Cells were mock infected, infected with MHV, or incubated with an equal concentration of UV-inactivated or heat-inactivated MHV and incubated with nanoparticles for 2-h intervals, beginning at infection (Fig. 2C). UV-inactivated MHV did not result in increased nanoparticle uptake, suggesting that surface receptor interactions were insufficient to induce macropinocytosis. Heat-inactivated MHV also did not cause nanoparticle internalization, suggesting that the membrane-ruffling phenotype observed was not a cellular response to foreign particles. In addition, in order to confirm that macropinocytosis is not utilized for entry of CoVs, we labeled virions with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and added them to DBT cells grown on a glass bottom dish at a high multiplicity of infection (MOI) of 25 PFU/cell (Fig. 2D). Virions were adsorbed for 30 min at 4°C to synchronize the infection before they were transferred to the 37°C chamber incubator surrounding the microscope. Cells were then live imaged for 1 h. While we did visualize viruses entering cells at the plasma membrane, we did not observe any evidence of cellular membrane ruffling at the site of entry or elsewhere on the cell. Thus, virus infection is required for induction of bulk fluid uptake, and MHV-induced macropinocytosis is not associated with virus entry.

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