Foot-and-mouth disease virus, but not bovine enterovirus, targets the host cell cytoskeleton via the nonstructural protein 3Cpro

Abstract

Foot-and-mouth disease virus (FMDV), a member of the Picornaviridae, is a pathogen of cloven-hoofed animals and causes a disease of major economic importance. Picornavirus-infected cells show changes in cell morphology and rearrangement of cytoplasmic membranes, which are a consequence of virus replication. We show here, by confocal immunofluorescence and electron microscopy, that the changes in morphology of FMDV-infected cells involve changes in the distribution of microtubule and intermediate filament components during infection. Despite the continued presence of centrosomes in infected cells, there is a loss of tethering of microtubules to the microtubule organizing center (MTOC) region. Loss of labeling for gamma-tubulin, but not pericentrin, from the MTOC suggests a targeting of gamma-tubulin (or associated proteins) rather than a total breakdown in MTOC structure. The identity of the FMDV protein(s) responsible was determined by the expression of individual viral nonstructural proteins and their precursors in uninfected cells. We report that the only viral nonstructural protein able to reproduce the loss of gamma-tubulin from the MTOC and the loss of integrity of the microtubule system is FMDV 3C(pro). In contrast, infection of cells with another picornavirus, bovine enterovirus, did not affect gamma-tubulin distribution, and the microtubule network remained relatively unaffected.

FIG. 1.

CPE in FMDV-infected cells. Still images from a live-cell experiment in which BHK-21 cells were infected with FMDV and imaged in a confocal microscope by DIC optics are shown. The three still images are from the cells at 0 (a), 76 (b), and 152 min (c) postinfection. Scale bar (for all three panels), 20 μm.

FIG. 2.

Effect of FMDV and BEV infection on host cell vimentin. BHK-21 cells were infected with FMDV and fixed with paraformaldehyde at 2 hpi (panels a and b, merged in panel c) and BEV fixed at 4 hpi (panels d and e, merged in panel f). An uninfected cell is shown in panel g. The cells were immunolabeled for the presence of viral structural proteins detected with anti-rabbit antibody-Alexa488 (green) and mouse anti-vimentin detected with anti-mouse antibody-Alexa568 conjugate (red). Nuclei were labeled with DAPI (blue). Scale bars: a to c, 10 μm; d to f, 20 μm; g, 10 μm.

FIG. 3.

Effect of FMDV and BEV infection on host cell microtubules. BHK-21 cells were infected with FMDV and fixed with paraformaldehyde at 2 hpi (panels a and b, merged in panel c) and BEV fixed at 4 hpi (panels d and e, merged in panel f). An uninfected cell is shown in panel g. The cells were immunolabeled for the presence of FMDV and BEV structural proteins, which were detected with anti-rabbit antibody-Alexa488 conjugate (green), and the microtubule cytoskeleton was immunolabeled with mouse anti-α-tubulin, which was detected with anti-mouse antibody-Alexa568 conjugate (red). Nuclei were labeled with DAPI (blue). Scale bars: a to f, 10 μm; g, 10 μm.

FIG. 4.

FMDV, but not BEV, induces loss of γ-tubulin from the MTOC. BHK-21 cells were infected with FMDV and fixed with paraformaldehyde at 2 hpi (panels a and b, merged in panel c) and BEV fixed at 4 hpi (panels d and e merged in panel f). The cells were immunolabeled for the presence of viral structural proteins, which were detected with anti-rabbit antibody-Alexa488 conjugate (green), and γ-tubulin, which was detected with anti-mouse antibody-Alexa568 conjugate to show the location of the MTOC (red). Nuclei were labeled with DAPI (blue). Scale bar, 10 μm.

FIG. 5.

Effect of FMDV infection or nonstructural protein 3C^pro expression on the MTOC. (a to c) Uninfected BHK-21 cells fixed in paraformaldehyde and labeled for γ-tubulin (a) and pericentrin (b) (green; arrows) or for TEM showing centrioles (c). (d and e) BHK21 cells infected with FMDV and fixed in paraformaldehyde at 2 hpi and labeled for FMDV structural proteins detected with anti-rabbit antibody-Alexa568 conjugate (red) and either γ-tubulin (d) or pericentrin (e) (green). (f) BHK-21 cells infected with FMDV and fixed for TEM at 2 hpi. The cell cytoplasm contains typical FMDV-induced vesicles, but the centrioles are visible in the center of the image. (g) BHK-21 cells transfected with 2BV5 and immunolabeled with anti-V5 tag detected by anti-mouse IgG2a-Alexa568 (red) and γ-tubulin detected by anti-mouse IgG1-Alexa488 (green). (h and i) BSR cells transfected with pSKRH3C, which expresses the FMDV 3C^pro, and immunolabeled for 3C^pro (antibody 4E9) detected by anti-mouse IgG2b-Alexa568 (red) and with mouse anti-γ-tubulin (green). Nuclei were labeled with DAPI (blue). Scale bars: a, b, d, e, g, h, and i, 10 μm; c, 500 nm; f, 400 nm.

FIG. 6.

Effects of FMDV and cycloheximide on γ- and α-tubulin. (a) BHK-21 cells were infected with FMDV 0₁BFS strain. Samples were lysed at 0, 2, 3, and 4 hpi. Proteins were separated by SDS-PAGE, blotted, and analyzed for the presence of FMDV 2C, α-tubulin, and γ-tubulin. (b) Cycloheximide was added to cells at a concentration of 100 μg/ml. Cells were examined over the subsequent 4 h. The level of α-tubulin synthesis in cells was examined at 0, 1, 2, 3, and 4 h after the addition of cycloheximide, by radiolabeling with ³⁵S followed by immunoprecipitation. (c) γ-Tubulin levels at the MTOC were examined by confocal microscopy at the same time points as in panel b. (d) BSR cells transfected with pSKRH3C, which expresses the FMDV 3C^pro, and immunolabeled for 3C^pro (antibody 4E9) detected by anti-mouse IgG2b-Alexa488 (green) and with mouse anti-α-tubulin detected with Alexa568 (red). Scale bars, 10 μm.