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. 2002 May;76(10):5266-70.
doi: 10.1128/jvi.76.10.5266-5270.2002.

Association of the caveola vesicular system with cellular entry by filoviruses

Cyril J Empig  1 , Mark A Goldsmith
Affiliations

Association of the caveola vesicular system with cellular entry by filoviruses

Cyril J Empig et al. J Virol. 2002 May.

Abstract

The filoviruses Ebola Zaire virus and Marburg virus are believed to infect target cells through endocytic vesicles, but the details of this pathway are unknown. We used a pseudotyping strategy to investigate the cell biology of filovirus entry. We observed that specific inhibitors of the caveola system, including cholesterol-sequestering drugs and phorbol esters, inhibited the entry of filovirus pseudotypes into human cells. We also measured slower cell entry kinetics for both filovirus pseudotypes than for pseudotypes of vesicular stomatitis virus (VSV), which has been recognized to exploit the clathrin-mediated entry pathway. Finally, visualization by immunofluorescence and confocal microscopy revealed that the filovirus pseudotypes colocalized with the caveola protein marker caveolin-1 but that VSV pseudotypes did not. Collectively, these results provide evidence suggesting that filoviruses use caveolae to gain entry into cells.

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Figures

FIG. 1.
FIG. 1.
Characterization of entry into human 293T cells by EBO-Z and MBG pseudotypes. Shown are the levels of infections in 293T cells treated with nystatin (25 μg/ml) or MβCD (500 μM) (A) and with PMA (10 μM) (B). Values are mean levels (± standard errors) of luciferase activity in cell lysates (n = 3). Dashed lines indicate infection of untreated control set at 100% infection. (C) Kinetics of pseudotype entry into 293T cells. The indicated pseudotypes were preadsorbed to 293T cells on ice for 30 min, and then the cells were shifted to 37°C. At 0, 0.5, 1, 2, 3, 4, 5, and 6 h following the shift, the cells were treated with trypsin (0.25%) to remove noninternalized virus. Infection was then measured 3 days postinfection by measuring luciferase activity in cell lysates. Values are mean levels (± standard errors) of luciferase activity in cell lysates (n = 3 for each time point). The horizontal dashed line indicates a 50% level of infection relative to the maximum level of infection at 6 h.
FIG. 2.
FIG. 2.
Inhibition of MBG infection of human HeLa cells. Shown are the levels of infection of HeLa cells treated with nystatin (50 μg/ml) (A), MβCD (5 mM) (B), and PMA (10 μM) (C). Values are mean levels (± standard errors) of luciferase activity in cell lysates (n = 3). The dashed lines indicate infection of untreated control set at 100% infection.
FIG. 3.
FIG. 3.
Colocalization of EBO-Z and MBG pseudotypes with the caveola protein marker CAV-1 after infection of HeLa cells. (A to I) Images of cells with Ctxn B-FITC (A to C), tfn-FITC (D to F), and the VSV, EBO-Z, and MBG pseudotypes (G, H, and I, respectively); (C) Ctxn B-FITC and CAV-1 colocalization following incubation of cells with Ctxn B-FITC for 30 min at 37°C; (F) absence of colocalization of tfn-FITC and CAV-1 following incubation of cells with tfn-FITC for 30 min at 37°C; (G) absence of colocalization of VSV and CAV-1 following incubation of cells with VSV pseudotypes for 30 min at 37°C; (H and I) colocalization of EBO-Z and MBG with CAV-1 following a 90-min incubation of cells with EBO-Z (H) and MBG (I) pseudotypes at 37°C. Following the incubations, all samples were fixed in paraformaldehyde (2%), permeabilized in Triton X-100 (0.1%), and immunostained for p24 (to detect pseudotypes) and/or CAV-1 (to detect caveolae). p24 was detected with an FITC-conjugated mouse anti-p24 antibody (Coulter Corp.), and the FITC signal was amplified with Alexa sandwich antibodies (Alexa Fluor 488-conjugated goat polyclonal anti-FITC antibody and Alexa Fluor 488-conjugated donkey anti-goat IgG (H+L) conjugate (Molecular Probes, Inc.). CAV-1 was detected with a rabbit polyclonal antibody against CAV-1 (BD Transduction Laboratories) and a Texas Red-conjugated anti-rabbit IgG (H+L) F(ab′)2 fragment (Jackson ImmunoResearch Laboratories, Inc.). Cell images were taken under oil at a ×60 magnification with a Bio-Rad Radiance2100 laser scanning confocal microscope system operated via LaserSharp2000 software. For intracellular analyses of cells, the z-axis position was computer controlled and the basal and apical membranes were set as the start and stop points, respectively. All images were at least 2 μm from either vantage point. Immunostained cells were excited with krypton or argon lasers at 488 nm (Alexa Fluor 488 and FITC) and 568 nm (Texas Red), and fluorescence signals were collected with barrier filters for Alexa Fluor 488 and FITC and for Texas Red. Regions of colocalization were generated by digital overlay with Photoshop 6 (Adobe Systems) and appear yellow (C and F to I). For comparison purposes, images collected with green and red barrier filters were digitally colorized to green (A and D) and red (B and E), respectively, also with Photoshop 6.
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