Coronaviruses induce entry-independent, continuous macropinocytosis

Abstract

Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV infection has not been investigated. We demonstrate that the CoVs SARS CoV and murine hepatitis virus (MHV) induce macropinocytosis, which occurs late during infection, is continuous, and is not associated with virus entry. MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells. MHV-induced macropinocytosis requires fusogenic spike protein on the cell surface and is dependent on epidermal growth factor receptor activation. Inhibition of macropinocytosis reduces supernatant viral titers and syncytia but not intracellular virus titers. These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading. Our studies are the first to demonstrate virus use of macropinocytosis for a role other than entry and suggest a much broader potential exploitation of macropinocytosis in virus replication and host interactions. Importance: Coronaviruses (CoVs), including severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV, are critical emerging human pathogens. Macropinocytosis is induced by many pathogens to enter host cells, but other functions for macropinocytosis in virus replication are unknown. 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. Murine hepatitis virus macropinocytosis requires a fusogenic virus spike protein and signals through the epidermal growth factor receptor and the classical macropinocytosis pathway. These studies demonstrate CoV induction of macropinocytosis for a purpose other than entry and indicate that viruses likely exploit macropinocytosis at multiple steps in replication and pathogenesis.

FIG 1

Infection with MHV or SARS CoV induces continuous membrane ruffling. (A) DBT cells were infected with MHV-Δ2-GFP3 at an MOI of 1 PFU/cell and imaged from 4 to 12 hpi. The small panels to the right are enlargements showing details of the white-boxed area. Green fluorescence indicates nsp3. Black arrowheads denote vesicles. White arrowheads follow the evolution of a vesicle over time. See Movie S1A to C in the supplemental material. (B) Vesicle diameter was measured in five movies of infected cells. MOI = 1 PFU/cell, n = 386 vesicles. (C, D) 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. Cells were fixed with 10% formalin and stained for F-actin (green). The arrow indicates a ruffle (C). Every third infected (or mock-infected) cell was imaged and scored for ruffling by three blinded reviewers. 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. n = ≥30 fields per replicate. *, P<0.05.

FIG 2

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.

FIG 3

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.

FIG 4

Inhibition of macropinocytosis impairs MHV replication. (A) Cells were reverse transfected for 68 h and infected with MHV for 12 h. The titers of supernatant samples were determined via plaque assay. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate. (B) The 12-h toxicity of CdtA was assessed with CellTiter-Glo. (C) Cells were treated with HEPES buffer or 10 µM CdtA for 2 h prior to infection with MHV A59 at an MOI of 1 PFU/cell. The viral titer was measured at 10 hpi. Data are represented as the mean ± the standard errors of the mean of an experiment done in triplicate. (D, E) Cells were infected with MHV at an MOI of 1 PFU/cell. At the times indicated postinfection, 0.4% DMSO or 10, 20, or 40 µM EIPA was added. The viral titer was measured at 12 hpi. EIPA was added at 6 hpi at 10, 20, or 40 µM (D) or at 2, 4, 6, or 8 hpi at 40 µM (E). Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. (F, G) Cells were infected with MHV-FFL2 at an MOI of 1 PFU/cell. At 8 hpi, 0.4% DMSO or 40 µM EIPA was added. Supernatant was collected at 10 hpi, and the viral titer was determined. Cells were collected in luciferase lysis buffer and assessed for luminescence (F) or in DMEM and subjected to three rounds of freezing and thawing before the titer was determined (G). Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n.s., not significant.

FIG 5

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.

FIG 6

MHV-induced macropinocytosis is associated with, but independent of, syncytium formation. (A) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell for 8 h, treated with PEG for 1 min, washed, and incubated for 3 h or transfected with VSV-G for 24 h. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Syncytia with ≤10 nuclei were analyzed. Data are represented as the mean ± the standard error of the mean of two replicates, each performed in duplicate. n = ≥30 cells per replicate. (B, C) Cells were mock infected or infected with MHV at an MOI of 1 PFU/cell. Anti-CEACAM blocking antibodies were added at 2 hpi, nanoparticles were added at 5 hpi, and cells were washed, fixed, stained, and imaged at 8 hpi. The number of nuclei per syncytium (B) and the percentage of cells with internalized nanoparticles (C) were measured. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. ***, P < 0.0001; **, P < 0.01; *, P < 0.05; n.s., not significant.

FIG 7

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.

FIG 8

Model of macropinocytosis during CoV infection. MHV 2S enters the cell via endocytosis after the spike protein interacts with the CEACAM receptor. Spike protein is cleaved to its fusogenic form by cathepsins after entry via endocytosis, and the virus fuses with the endosomal membrane to release the genome to the cytoplasm. Replication occurs, and virions are assembled in the ERGIC and then packaged and released via exocytosis. Spike protein that reaches the surface of the cell is uncleaved (black ball and stick) and cannot mediate syncytium formation with neighboring cells. MHV A59 enters the cell via fusion at the cell membrane after the spike protein interacts with the CEACAM receptor. The genome immediately enters the cytoplasm, and replication occurs. Packaging of nascent virions occurs in the ERGIC, and free spike proteins and spike protein incorporated into virions are cleaved by furin in the trans-Golgi compartment. Virions are packaged and released via exocytosis. Spike protein that reaches the cell surface is cleaved and fusogenically active (red ball and stick) and can mediate fusion events with neighboring cells. This cleaved, activated spike protein can also mediate interactions, potentially through the EGFR (purple ball and stick), that induce the macropinocytosis pathway within the cell, which relies on Rac1, Cdc42, and Pak1. Actin modifications at the cell surface then cause membrane ruffling and macropinosome internalization, in addition to filopodia that can facilitate spike protein-receptor interactions with neighboring cells. The location of spike protein cleavage is denoted by the red-striped regions.