Internalization of echovirus 1 in caveolae

Abstract

Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor alpha 2 beta 1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, alpha 2 beta 1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.

FIG. 1.

(A) Immunofluorescent labeling of EV1 using anti-EV1 rabbit hyperimmune serum in SAOS-α2β1 cells after viral attachment (0 h), 2 h p.i., or 8 h p.i. (Bars = 10 μm). (B) Low-magnification picture of anti-EV1 labeling in α2β1-negative vector control SAOS-pAW cells and in SAOS-α2β1 cells 10 h p.i. (Bars = 100 μm).

FIG. 2.

(A) Western blot of SAOS-pAW and SAOS-α2β1 cell homogenates without EV1 infection (C) or 0, 2, or 12 h p.i. (B) Binding of radioactively labeled EV1 onto SAOS-pAW and SAOS-α2β1 cells. (C) The proportional number of infected cells in SAOS-pAW and SAOS-α2β1 cells 10 h p.i. calculated after immunoperoxidase labeling. (D) The blocking effect of various antibodies on EV1 infection 10 h p.i. calculated after immunoperoxidase labeling (antibodies used in combination are 1950Z for anti-α2 and AB730 for anti-β2m). (E) Production of infectious EV1 in SAOS-α2β1 and SAOS-α2/α1β1 cells. The amount of intracellular virus in SAOS cells was determined in GMK cells as end point titers.

FIG. 3.

(A and B) Sucrose gradient sedimentation analysis of conformational changes in EV1 capsid structure during attachment and entry to the cells. After incubation of the SAOS-α2β1 cells with EV1 for 1 h on ice (A), they were transferred to 37°C for 2 h (B).

FIG. 4.

(A) Colocalization of α2β1 integrin (green) with EV1 (red) in SAOS-α2β1 cells after viral attachment on ice (0 h) or after 2 h of infection at 37°C. (B) Double-labeling with anti-β2 microglobulin and anti-α2 integrin antibodies 2 h p.i. Colocalization is visualized as the yellow color in merge images. Bar = 10 μm.

FIG. 5.

Double-labeling of EV1-infected SAOS-α2β1 cells with EV1 antiserum or anti-α2β1 integrin antibody together with markers connected to the clathrin-dependent endocytic route. EEA1/α2β1, α2β1 integrin (green) with the early endosomal marker EEA1 (red); TF-myc/EV1, EV1 capsid proteins (red) with the myc-tagged transferrin receptor, a marker for the early endosomes (green); CI-MPR/α2β1, α2β1 integrin (green) with late endosomal marker (red); TGN-46/α2β1, α2β1 integrin (green) with Golgi marker TGN-46 (red). Bar = 10 μm.

FIG. 6.

Electron micrographs of caveolae in SAOS-α2β1 cells. (A) Section of an uninfected cell showing several caveolae. (B to D) Immunolocalization of EV1 (5 nm proteinA-gold) in a caveola-like membrane invagination (B), in a cluster of caveolae deeper in the cytoplasm 30 min p.i. (C), and in a large accumulation close to the nucleus (n) 2 h p.i. (D). (E and F) Double cryo-immunolabeling of caveolin-1 (10-nm-diameter protein A-gold particles) and EV1 (5-nm-diameter protein A-gold particles) in a caveola-like structure close to the plasma membrane (5-nm-diameter protein A-gold particles are indicated by arrowheads). Bars = 100 nm.

FIG. 7.

Labeling of caveolin-1 in SAOS-α2β1 cells after viral attachment (A) and 2 h p.i. (B). (C) Double-labeling of caveolin-1 (green) with EV1 (red) after 2 h p.i. (D) Caveolin-1 (red) with α2β1 integrin (green) after 2 h p.i. Colocalization is visualized as a yellow color in merged images (C and D). Bar = 10 μm.

FIG. 8.

Immunoisolation of caveola structures with anti-caveolin-1 Dynal beads. Immunoisolation was performed in SAOS-α2β1 cells after viral attachment (0) or 15 min p.i. The Western blot has been stained with anti-EV1 antiserum. Dynal beads coated with nonspecific mouse IgG were used as control. Abbreviations: H, homogenate; B, bound fraction. UB, unbound fraction.