Cell entry of the aphthovirus equine rhinitis A virus is dependent on endosome acidification

Abstract

Equine rhinitis A virus (ERAV) is genetically closely related to foot-and-mouth disease virus (FMDV), and both are now classified within the genus Aphthovirus of the family Picornaviridae. For disease security reasons, FMDV can be handled only in high-containment facilities, but these constraints do not apply to ERAV, making it an attractive alternative for the study of aphthovirus biology. Here, we show, using immunofluorescence, pharmacological agents, and dominant negative inhibitors, that ERAV entry occurs (as for FMDV) via clathrin-mediated endocytosis and acidification of early endosomes. This validates the use of ERAV as a model system to study the mechanism of cell entry by FMDV.

FIG. 1.

ERAV internalization time course. ERAV-Cy2 was bound to cells in the cold, and unbound virus was washed away before infection initiated at physiological temperature. Cells were fixed at 0, 5, and 10 min pi (panels a, b, and c, respectively).

FIG. 2.

ERAV colocalization with endocytosis markers. Cells were infected with Cy2-labeled ERAV for 0 to 30 min before being visualized as described in the text. Antibodies against endocytosis markers were used in combination with secondary antibodies conjugated to Alexa-594 (Invitrogen). Cell nuclei were stained with Hoechst. Images were acquired using a DeltaVision deconvoluting microscope. (a) ERAV-Cy2 (green). (b) Clathrin (red) at 10 min pi. (c) Merge of panels a and b, showing colocalization (yellow) of ERAV and clathrin. (e to t) Cells fixed at 20 min pi with ERAV-Cy2 (green) and caveolin-1, Tfr-R, EEA1, or CD63 (red). (g) Overlay of panels e and f, showing ERAV (green) and caveolin-1 (red). (k and o) Colocalization (yellow) of ERAV with Tfr-R or EEA1, respectively. Panel k is a merge of panels i and j, and panel o is a merge of panels m and n. (t) Overlay of panels r and s, showing ERAV (green) and CD63 (red). Panels d, h, l, p, q, and u are enlargements from the Merge panels.

FIG. 3.

Effects of inhibition of clathrin-mediated endocytosis on ERAV entry. (A) Detection of ERAV (red) 20 min pi (b and f) of cells expressing wild-type Eps15 (Eps15-WT-GFP) (green) (a) or dominant negative Eps15 (Eps15-DN-GFP) (green) (e), which inhibits clathrin-mediated endocytosis. ERAV is located predominantly in the cytoplasm in cells expressing wild-type Eps15 (c, overlay of a and b). Panel d is an enlargement from panel c. ERAV is located predominantly at the cell surface in cells expressing dominant negative Eps15 (g, overlay of panels e and f). Panel h is an enlargement from panel g. (B) Panels i to o show cells fixed at 6 h pi. Panel k is a merge of panels i (Eps15-WT-GFP [green]) and j (ERAV [red]). The large ERAV signal in the cytoplasm is indicative of replication. Panel o is a merge of panels m (Eps15-DN-GFP [green]) and n (ERAV [red]). The lack of ERAV signal in the cytoplasm suggests lack of replication.

FIG. 4.

Effects of pharmacological inhibitors of clathrin- and caveolin-dependent endocytosis and endosomal acidification on ERAV infectivity. (A) HeLa Ohio cells were treated with inhibitors of clathrin-dependent endocytosis and then infected with ERAV. The number of virus plaques generated in the presence of the inhibitors was expressed as a percentage of the no-drug control. ERAV infectivity was inhibited 40% by chlorpromazine (Cpz; 15 μM) and 85% by sucrose (Sucr; 0.4 M). (B) Effects of inhibition of caveolin-dependent endocytosis on ERAV. Neither nystatin (Nys) nor MβCD reduced the number of plaques compared to untreated controls. (C) Cells were treated with concanamycin A (ConcA) (b) or dimethyl sulfoxide (DMSO) alone (a) for 30 min at 37°C followed by visualization of endosomal acidification by AO fluorescence. In mock-treated cells, the acidic vesicles appear orange due to accumulation of AO, while in concanamycin A-treated cells, no red/orange color is detected, confirming inhibition of endosomal acidification (see the text). (D) Reduction of ERAV infectivity by inhibition of endosomal acidification with concanamycin A (100 nm) or monensin (Mon; 25 μM). (E) Progression from early to late endosomes is not necessary for ERAV infectivity since nocodazole (Noc; 20 μM) or wortmannin (Wort; 25 nM) did not reduce plaque numbers.