Rotavirus spike protein VP4 binds to and remodels actin bundles of the epithelial brush border into actin bodies

Abstract

We demonstrate here that VP4, a rotaviral protein, is able to specifically bind to bundled actin microfilaments that are subsequently profoundly remodeled into actin bodies. These cytoplasmic actin bodies do not localize within identified intracellular compartments. VP4-induced actin remodeling is similar to cytochalasin D effects with kinetics compatible with that of rotavirus infection. Actin bundles' remodeling occurs both in infected and in VP4-transfected cells and in various cell lines, indicating that this is a general property of the viral protein itself. Interestingly, in intestinal epithelial cells, which represent the natural target of rotavirus, VP4 is addressed to the apical membrane where it binds specifically to brush border actin bundles and elicits its remodeling, whereas cytochalasin D impaired all the filamentous actin. These observations indicate that these original properties of VP4 likely explain the previously described brush border alterations that follow rotavirus infection of enterocytes and may also participate to the mechanism of rotavirus final assembly.

FIG. 1.

VP4, the spike rotavirus protein, localizes within filaments and dots in infected and transfected Cos-7 cells. Cos-7 cells were infected with the rotavirus at 1 PFU/cell for 4 h (A) or transiently transfected with plasmids encoding VP4 (B) or EGFP-VP4 (C) for 24 h. Cells were then processed for VP4 staining (A and B). Images gallery displays projections of all 0.5-μm xy focal sections taken throughout the height of the cells. Scale bars = 10 μm.

FIG. 2.

EGFP-VP4 specifically colocalizes with polymerized actin. Cos-7 cells were analyzed after 24 h pEGFP-VP4 transient transfection. EGFP-VP4 expressing cells (A, E, I, and L) were immunostained with anti-α-tubulin (C and N), antivimentin (G), or anticytokeratin (J) and/or colabeled with phalloidin to detect F-actin (B, F, and M). EGFP-VP4 expressing Cos-7 cells in panels L through O were treated for 1 h with 10 μM of cytochalasin D before fixation. Images gallery displays confocal single xy planes. Note that colabeling of anticytokeratin and phalloidin is not possible because methanol fixation for cytokeratin preservation is not compatible with phalloidin labeling. Scale bars = 10 μm.

FIG. 3.

VP4 and actin interact in infected Cos-7 cells. Cos-7 cells were infected with rotavirus at 1 PFU/cell for 4 h (A through C), with 1 h of 10 μM cytochalasin D treatment (D through F). Cells were then processed for phalloidin labeling (B and E) and VP4 immunostaining (A and D). Images gallery displays confocal single xy planes. Scale bars = 10 μm. Lysates from mock-infected or 4-h-infected Cos-7 cells were subjected to immunoprecipitation with anti-VP4 antibodies and then to Western blotting using anti-VP4 and antiactin (G). Lane a, mock-infected Cos-7 cells lysates immunoprecipitated with anti-VP4 antibodies. Lanes b and c, infected Cos-7 cells lysates immunoprecipitated with nonrelevant antibody and anti-VP4 antibodies, respectively. Note that immunoglobulins were not detected because anti-VP4 was covalently linked to protein A-Dynabeads.

FIG. 4.

EGFP-VP4 does not localize within identified vesicular compartments. Cos-7 cells were fixed after 24-h pEGFP-VP4-transient transfection, permeabilized, and vesicular compartment markers were stained by indirect immunofluorescence. The major compartments of the exocytic and endocytic pathways were explored using antibodies against protein disulfide isomerase for the endoplasmic reticulum, ERGIC-53 for the intermediate compartment, giantin for the Golgi apparatus, TGN46 protein for the trans-Golgi network, the early endosomal antigen-1 for early endosomes, transferrin receptor for late endosomes, lysotracker for lysosomes, Mitotracker for mitochondria, and either anticatalase antibodies or a dsRed-labeled plasmid containing the peroxisome targeting signal PTS1 for peroxisomes. EGFP-VP4-expressing cells (A′, B′, C′, D′, E′, F′, G′, H′, I′, and J′) were costained with anti-protein disulfide isomerase (PDI) (A”), anti-ERGIC-53 (B"), anti-giantin (C"), anti-TGN-46 (D"), anti-early endosome antigen 1 (E"), anti-transferrin receptor (F"), lysotracker (G"), mitotracker (H"), or anti-catalase (I"). (J′) displays Cos-7 cells cotransfected with plasmids encoding EGFP-VP4 and PST1-dsRed, respectively. Images gallery displays confocal single xy planes. Scale bars = 10 μm.

FIG. 5.

VP4-induced actin remodeling partially mimics cytochalasin D. Control EGFP transfected Cos-7 cells were labeled with Alexa 547-phalloidin at 48 h posttransfection (A through C). Cos-7 cells expressing EGFP-VP4 were analyzed 24 h (D through G), 48 h (H through K) or 72 h (L through O) posttransfection. These transfected cells (A, D, H, and L) or cells treated with cytochalasin D (10 μM) for 1 h (P through R) or for 3 h (S through U) were stained using Alexa 547-phalloidin (B, E, I, M, P, and S) or anti-α-tubulin antibody (F, J, N, Q, and T). Images gallery displays projections of all 0.5-μm xy focal sections taken throughout the height of cells. Scale bars = 10 μm.

FIG. 6.

VP4 remodels actin microfilaments into actin bodies. EGFP-VP4 expressing Cos-7 cells were mounted in a POC chamber and cells were perfused with 37°C 50 mM HEPES and OPTI-MEM medium. Images were acquired with a confocal microscope from time zero (To) to 15 min after perfusion (T+15min) every 3 min. Image gallery displays projections of all 0.3-μm xy focal sections taken throughout the height of a zoomed region of a EGFP-VP4-expressing cell. Scale bar = 10 μm.

FIG. 7.

VP4 localizes at the brush border and perturbs actin bundles organization. EGFP-VP4 expressing Caco-2 cells were labeled 72 h after nucleofection with Alexa 547-phalloidin. Image gallery displays an apical plane these cells (A) or xz section (B). Fifteen-day-old Caco-2 cells were infected with rotavirus for 6 h (C through G) or for 18 h (H and I). A 1-h cytochalasin D (cytoD) (10 μM) treatment was performed after at 5 h p.i. (F and G). Cells were then processed for Alexa 547-phalloidin labeling and VP4 immunostaining using MAb 7.7. Images gallery displays apical planes (A, C, F, and H), basal planes (D and G) or xz sections (B, E, and I). Scale bar = 5 μm.

FIG. 8.

Actin is coimmunoprecipitated with VP4 from 18-h-infected Caco-2 cells. Differentiated Caco-2 cells were infected with rotavirus at 10 PFU/cell. Lysates from mock-infected or 18-h-infected Caco-2 cells were subjected to immunoprecipitation with anti-VP4 antibodies and then to Western blotting using anti-VP4 and anti-actin. Lane a, mock-infected Caco-2 cells lysates immunoprecipitated with anti-VP4 antibodies; lanes b and c, infected Caco-2 cells lysates immunoprecipitated with nonrelevant antibody and anti-VP4 antibodies, respectively. Note that immunoglobulins were not detected because anti-VP4 was covalently linked to protein A-Dynabeads.