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. 2005 Jul;79(13):8506-18.
doi: 10.1128/JVI.79.13.8506-8518.2005.

Analysis of foot-and-mouth disease virus internalization events in cultured cells

Vivian O'Donnell  1 , Michael LaRocco, Hernando Duque, Barry Baxt
Affiliations

Analysis of foot-and-mouth disease virus internalization events in cultured cells

Vivian O'Donnell et al. J Virol. 2005 Jul.

Abstract

It has been demonstrated that foot-and-mouth disease virus (FMDV) can utilize at least four members of the alpha(V) subgroup of the integrin family of receptors in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the betaG-betaH loop of VP1. While there have been extensive studies of virus-receptor interactions at the cell surface, our understanding of the events during viral entry into the infected cell is still not clear. We have utilized confocal microscopy to analyze the entry of two FMDV serotypes (types A and O) after interaction with integrin receptors at the cell surface. In cell cultures expressing both the alphaVbeta3 and alphaVbeta6 integrins, virus adsorbed to the cells at 4 degrees C appears to colocalize almost exclusively with the alphaVbeta6 integrin. Upon shifting the infected cells to 37 degrees C, FMDV capsid proteins were detected within 15 min after the temperature shift, in association with the integrin in vesicular structures that were positive for a marker of clathrin-mediated endocytosis. In contrast, virus did not colocalize with a marker for caveola-mediated endocytosis. Virus remained associated with the integrin until about 1 h after the temperature shift, when viral proteins appeared around the perinuclear region of the cell. By 15 min after the temperature shift, viral proteins were seen colocalizing with a marker for early endosomes, while no colocalization with late endosomal markers was observed. In the presence of monensin, which raises the pH of endocytic vesicles and has been shown to inhibit FMDV replication, viral proteins were not released from the recycling endosome structures. Viral proteins were not observed associated with the endoplasmic reticulum or the Golgi. These data indicate that FMDV utilizes the clathrin-mediated endocytosis pathway to infect the cells and that viral replication begins due to acidification of endocytic vesicles, causing the breakdown of the viral capsid structure and release of the genome by an as-yet-unidentified mechanism.

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Figures

FIG. 1.
FIG. 1.
Distribution of αVβ3 and β6 integrins in MCF-10A cells. Monolayers of uninfected MCF-10A cells were processed for IF staining as described in Materials and Methods. MAbs LM609, which recognizes the αVβ3 heterodimer (a), and CSβ6, which recognizes the β6 integrin subunit (b), were used as primary antibodies. Alexa Fluor 488-conjugated antibodies were used as secondary antibodies. The bars represent 16 μm in panel a and 20 μm in panel b.
FIG. 2.
FIG. 2.
FMDV replication in MCF-10A cells. Monolayers of MCF-10A and BHK-21 cells were infected with type A12 (a) or O1C (b) at an MOI of 10 PFU/cell for 1 h at 37°C. After the adsorption period, the cells were washed with MES-buffered saline (see Materials and Methods) and incubated at 37°C. At the times indicated, the plates were removed to −70°C. Samples were thawed, and titers were determined by plaque assay on BHK-21 cells. Parallel cultures of MCF-10A cells were processed for IF-confocal microscopy as described in Materials and Methods at the times indicated after the adsorption period (bottom). Viral protein synthesis was visualized with a specific MAb against type A12 or type O1C VP1. Anti-mouse isotype-specific IgG Alexa Fluor 594-labeled conjugate was used as a secondary antibody. The bars represent 8 μm.
FIG. 3.
FIG. 3.
Distribution of FMDV virions and integrin receptors during the viral adsorption period. Monolayers of MCF-10A cells were infected with FMDV type A12 or O1C at an MOI of 100 PFU/cell for 1 h at 4°C and processed for double IF staining as described in Materials and Methods. FMDV virions were localized with specific MAbs against the capsid protein VP1 and visualized with Alexa Fluor 594 (red). Integrins were stained with anti-αVβ3 (a) or anti-β6 (b) MAbs and visualized with Alexa Fluor 488 (green). The bars represent 20 μm in panel a and 8 μm in panel b.
FIG. 4.
FIG. 4.
Internalization of FMDV and αVβ6. FMDV type O1C was adsorbed to MCF-10A monolayers at an MOI of 100 PFU/cell for 1 h at 4°C. The cells were washed with medium and incubated at 37°C. At the times indicated after the temperature shift, the cells were processed for IF-confocal microscopy as described in Materials and Methods. Virus was stained with an anti-VP1 MAb and visualized with Alexa Fluor 594 (red), and the αVβ6 integrin was stained with an anti-β6 MAb and visualized with Alexa Fluor 488 (green). The bars represent 8 μm.
FIG. 5.
FIG. 5.
Analysis of the FMDV internalization pathway. FMDV types A12 and O1C were adsorbed to monolayers of MCF-10A cells (MOI, 100 PFU/cell) for 1 h at 4°C. The cells were washed, overlaid with warm medium, and transferred to 37°C. At the times indicated after the temperature shift, cells were processed for confocal microscopy as described in Materials and Methods. Virus was stained with anti-VP1 MAbs and visualized with Alexa Fluor 594 (red). Clathrin (a) was stained with an anti-clathrin MAb, and caveolin-1 (b) was stained with a rabbit polyclonal anti-caveolin-1 antibody. Both proteins were visualized with Alexa Fluor 488 (green). Only the merged photographs are shown. In panel c, cells were pretreated with chlorpromazine (Cpz; 12.5 μM) for 30 min at 37°C prior to infection with type O1C (MOI, 100 PFU/cell). The cells were incubated at 37°C in the presence of the drug until 4 h postinfection, when they were processed for confocal microscopy. Virus was stained with an anti-VP1 MAb and visualized with Alexa Fluor 594 (red). The bars represent 8 μm in panels a and b and 40 μm in panel c.
FIG. 6.
FIG. 6.
Movement of virus into early endosomes. Monolayers of COS-1 cells were cotransfected with cDNA plasmids encoding the bovine αV and β6 subunits as described in Materials and Methods. At 24 h posttransfection, cells were infected with type O1C (MOI, 100 PFU/cell) for 1 h at 4°C. After being washed, the cells were overlaid with warm medium and moved to 37°C. At the times indicated after the temperature shift, cells were processed for confocal microscopy. Virus was stained with an anti-VP1 MAb and visualized with Alexa Fluor 594 (red). Early endosomes were stained with a rabbit polyclonal anti-EEA-1 antiserum and visualized with Alexa Fluor 488 (green). The bars represent 8 μm.
FIG. 7.
FIG. 7.
Interaction of FMDV with late endosomes. Monolayers of MCF-10A cells were infected with type A12 (MOI, 100 PFU/cell) for 1 h at 4°C. After being washed, the cells were overlaid with warm medium and moved to 37°C. At the times indicated after the temperature shift, cells were processed for confocal microscopy. Virus was stained with an anti-VP1 MAb and visualized with Alexa Fluor 594 (red). Late endosomes were stained with a rabbit polyclonal anti-CI-MPR antiserum and visualized with Alexa Fluor 488 (green). The bars represent 8 μm.
FIG. 8.
FIG. 8.
Effect of monensin on virus internalization. Monolayers of MCF-10A cells were incubated with monensin (50 μM) for 30 min at 37°C prior to infection with type O1C (a) or type A12 (b) (MOI, 100 PFU/cell) for 1 h at 4°C. After being washed, the cells were overlaid with warm medium in the presence of monensin and moved to 37°C. At 4 h p.a., cells were processed for confocal microscopy. Virus was stained with an anti-VP1 MAb and visualized with Alexa Fluor 594 (red). The integrin αVβ6 was stained with an anti-β6 MAb (a), and the TfnR was stained with an anti-TfnR MAb (b). Both proteins were visualized with Alexa Fluor 488 (green). The bars represent 8 μm.
FIG. 9.
FIG. 9.
Interaction of FMDV with ER and Golgi apparatus. Monolayers of MCF-10A cells were infected with type O1C (MOI, 100 PFU/cell) for 1 h at 4°C. After being washed, the cells were overlaid with warm medium and moved to 37°C. At the times indicated after the temperature shift, cells were processed for confocal microscopy. Virus was stained with an anti-VP1 MAb and visualized with Alexa Fluor 594 (red). The ER was stained with an anti-PDI MAb, and the Golgi was stained with an anti-Golgi zone area MAb. Both proteins were visualized with Alexa Fluor 488 (green), and only the merged photographs are shown. The bars represent 8 μm.
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