Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses

Abstract

Spatiotemporal aspects of filovirus entry and release are poorly understood. Lipid rafts act as functional platforms for multiple cellular signaling and trafficking processes. Here, we report the compartmentalization of Ebola and Marburg viral proteins within lipid rafts during viral assembly and budding. Filoviruses released from infected cells incorporated raft-associated molecules, suggesting that viral exit occurs at the rafts. Ectopic expression of Ebola matrix protein and glycoprotein supported raft-dependent release of filamentous, virus-like particles (VLPs), strikingly similar to live virus as revealed by electron microscopy. Our findings also revealed that the entry of filoviruses requires functional rafts, identifying rafts as the site of virus attack. The identification of rafts as the gateway for the entry and exit of filoviruses and raft-dependent generation of VLPs have important implications for development of therapeutics and vaccination strategies against infections with Ebola and Marburg viruses.

Figure 3.

Localization of filovirus proteins in lipid rafts in infected cells. (A) Primary human monocytes were infected with MBGV. After 24 h cells were lysed in 0.5% Triton X100 and detergent-soluble (S) and -insoluble (I) fractions were separated by centrifugation, samples were irradiated (2 × 10⁶ R), and analyzed by immunoblotting with a guinea pig anti-MBGV antibody to detect viral proteins NP and VP35/VP40 (lanes 3 and 4); lanes 1 and 2, uninfected control; lane 5, inactivated MBGV (1 μg). NS, nonspecific band. (B) HepG2 hepatocytes were infected with EBOV-Zaire, lysed, irradiated (6 × 10⁶ R), and rafts (R) and soluble (S) fractions were prepared by ultracentrifugation 24 h after infection. Ebola GP and VP40 were detected by immunoblotting. (C) Ebola-infected Vero E6 cells were irradiated (4 × 10⁶ R), fixed and stained for Ebola virus (red) and GM1 (green) at 4°C and imaged by confocal microscopy. (Left panel) single section; (right panel), 3-D reconstruction of the compiled data.

Figure 3.

Localization of filovirus proteins in lipid rafts in infected cells. (A) Primary human monocytes were infected with MBGV. After 24 h cells were lysed in 0.5% Triton X100 and detergent-soluble (S) and -insoluble (I) fractions were separated by centrifugation, samples were irradiated (2 × 10⁶ R), and analyzed by immunoblotting with a guinea pig anti-MBGV antibody to detect viral proteins NP and VP35/VP40 (lanes 3 and 4); lanes 1 and 2, uninfected control; lane 5, inactivated MBGV (1 μg). NS, nonspecific band. (B) HepG2 hepatocytes were infected with EBOV-Zaire, lysed, irradiated (6 × 10⁶ R), and rafts (R) and soluble (S) fractions were prepared by ultracentrifugation 24 h after infection. Ebola GP and VP40 were detected by immunoblotting. (C) Ebola-infected Vero E6 cells were irradiated (4 × 10⁶ R), fixed and stained for Ebola virus (red) and GM1 (green) at 4°C and imaged by confocal microscopy. (Left panel) single section; (right panel), 3-D reconstruction of the compiled data.

Figure 2.

Colocalization of filovirus GPs with GM1 on intact cells. (A) 293T cells were transfected with the indicated GP, and stained at 4°C with Alexa488-CTB (green) and anti-GP mAb followed by Alexa-647–conjugated anti–mouse antibodies (red), cells were fixed and imaged using confocal microscopy. Colocalization is represented by yellow appearance in the overlay (right panels). A 3-D reconstruction of the compiled data from 25 sections of an Ebo-GP–transfected cell is also shown. (B) 293T cells were concurrently stained at 4°C with Alexa-488–conjugated anti-TrfR antibody (green) and rhodamine-CTB (red), fixed and analyzed by confocal microscopy. No colocalization between these two molecules was observed, evident by the lack of yellow appearance in the overlay. Two representative cells are shown.

Figure 1.

Localization of filovirus GPs in lipid rafts. 293T cells were transfected with Marburg GP (A), Ebo-GPwt, or Ebo-GP_C670/672A (B), or a control plasmid, rafts were prepared by ultracentrifugation and GP was detected by immunoblotting. GM1 was detected by blotting with HRP-CTB in the corresponding fractions spotted on a nitrocellulose membrane, as a control for the quality of raft preparation. (C) 48 h after transfection of 293T cells with Ebola GP, a portion of cells were treated for 20 min with 10 mM methyl-β-cyclodextrin (MβCD) and another portion was left untreated. Raft and soluble fractions were prepared and analyzed by immunoblotting for GP (top panel) and for the raft-excluded protein TrfR (bottom panel).

Figure 1.

Localization of filovirus GPs in lipid rafts. 293T cells were transfected with Marburg GP (A), Ebo-GPwt, or Ebo-GP_C670/672A (B), or a control plasmid, rafts were prepared by ultracentrifugation and GP was detected by immunoblotting. GM1 was detected by blotting with HRP-CTB in the corresponding fractions spotted on a nitrocellulose membrane, as a control for the quality of raft preparation. (C) 48 h after transfection of 293T cells with Ebola GP, a portion of cells were treated for 20 min with 10 mM methyl-β-cyclodextrin (MβCD) and another portion was left untreated. Raft and soluble fractions were prepared and analyzed by immunoblotting for GP (top panel) and for the raft-excluded protein TrfR (bottom panel).

Figure 4.

Incorporation of GM1 in released filovirus virions. (A) Ebola virus was immunoprecipitated from supernatant of infected Vero-E6 cells (lane 2), or uninfected cells as control (lane 1), using anti-GP mAb. After irradiation (2 × 10⁶ R), a fraction of immunoprecipitate (IP) was spotted on nitrocellulose membrane and blotted with HRP-conjugated CTB to detect GM1 (bottom panel). Another portion of the IP was analyzed by SDS-PAGE and immunoblotting with anti-GP mAb (top panel). (B) MBGV (1 μg), prepared by ultracentrifugation and inactivated by radiation (10⁷ R), was analyzed for the presence of GM1, TrfR, and GP in a similar fashion. As control, rafts and soluble fractions from untransfected 293T cells were used.

Figure 4.

Incorporation of GM1 in released filovirus virions. (A) Ebola virus was immunoprecipitated from supernatant of infected Vero-E6 cells (lane 2), or uninfected cells as control (lane 1), using anti-GP mAb. After irradiation (2 × 10⁶ R), a fraction of immunoprecipitate (IP) was spotted on nitrocellulose membrane and blotted with HRP-conjugated CTB to detect GM1 (bottom panel). Another portion of the IP was analyzed by SDS-PAGE and immunoblotting with anti-GP mAb (top panel). (B) MBGV (1 μg), prepared by ultracentrifugation and inactivated by radiation (10⁷ R), was analyzed for the presence of GM1, TrfR, and GP in a similar fashion. As control, rafts and soluble fractions from untransfected 293T cells were used.

Figure 5.

Release of Ebola GP and VP40 as GM1-containing particles. (A) 293T cells were transfected with the indicated plasmids, supernatants were cleared from floating cells by centrifugation, and particulate material was pelleted through 30% sucrose by ultracentrifugation. The individual proteins were detected in the cell lysates and in the particulate material from supernatant by immunoblotting (IB). A fraction of cleared supernatant was subjected to immunoprecipitation using anti-GP mAb and analyzed for the presence of GM1 (bottom panel) as described in the legend to Fig. 1. (B) The particulate material from cells transfected with GP+VP40 was further purified on a sucrose step gradient and the low density fraction was analyzed for the presence of VP 40 (top panel), TrfR (middle panel), and GM1 (bottom panel). Rafts and soluble fractions from untransfected 293T cells were used as control.

Figure 5.

Release of Ebola GP and VP40 as GM1-containing particles. (A) 293T cells were transfected with the indicated plasmids, supernatants were cleared from floating cells by centrifugation, and particulate material was pelleted through 30% sucrose by ultracentrifugation. The individual proteins were detected in the cell lysates and in the particulate material from supernatant by immunoblotting (IB). A fraction of cleared supernatant was subjected to immunoprecipitation using anti-GP mAb and analyzed for the presence of GM1 (bottom panel) as described in the legend to Fig. 1. (B) The particulate material from cells transfected with GP+VP40 was further purified on a sucrose step gradient and the low density fraction was analyzed for the presence of VP 40 (top panel), TrfR (middle panel), and GM1 (bottom panel). Rafts and soluble fractions from untransfected 293T cells were used as control.

Figure 6.

Electron microscopic analysis of VLPs generated by EBOV GP and VP40. Particles obtained by ultracentrifugation of the supernatants of 293T cells transfected with Ebola GP+VP40 were negatively stained with uranyl-acetate to reveal the ultrastructure (A), or stained with anti-EBOV-GP mAb followed by Immunogold rabbit anti–mouse Ab (B), and analyzed by electron microscopy. The length of each particle is indicated in μm. (C) 293T cells transfected with Ebola GP+VP40 were immunogold-stained for Ebola GP, fixed, cut, and analyzed by electron microscopy. The picture depicts a typical site of VLP release from the cells, indicated by arrows. A magnification of the site of VLP release is also shown to better visualize the gold staining on the particles.

Figure 6.

Electron microscopic analysis of VLPs generated by EBOV GP and VP40. Particles obtained by ultracentrifugation of the supernatants of 293T cells transfected with Ebola GP+VP40 were negatively stained with uranyl-acetate to reveal the ultrastructure (A), or stained with anti-EBOV-GP mAb followed by Immunogold rabbit anti–mouse Ab (B), and analyzed by electron microscopy. The length of each particle is indicated in μm. (C) 293T cells transfected with Ebola GP+VP40 were immunogold-stained for Ebola GP, fixed, cut, and analyzed by electron microscopy. The picture depicts a typical site of VLP release from the cells, indicated by arrows. A magnification of the site of VLP release is also shown to better visualize the gold staining on the particles.

Figure 7.

Inhibition of Ebola infection by raft-disrupting agents filipin and nystatin. Vero E6 cells were left untreated or treated for 30 min with 0.2 μg/ml of filipin or 100 U/ml of nystatin at 37°C, washed extensively with PBS, and infected with EBOV at an MOI of 1. As a control for lack of general toxicity and persistent effect on viral replication, upon treatment with Filipin, cells were washed, and incubated in medium for 4 h before infection with EBOV (Filipin→recovery). After 48 h supernatants were harvested and viral titers determined by plaque assay.