The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment

Abstract

Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38 degrees C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.

FIG. 1

Apical binding and endocytosis of TGEV by pAPN-MDCK cells. (A) A large number of TGEV particles are seen in close proximity to the apical membrane after initial adsorption (60 min, 4°C) and incubation at 38°C (3 min). (B and C) TGEV particles (arrows) are seen in pits with a thickened plasma membrane after adsorption and incubation at 38°C for 3 min. (D) TGEV particle in a smooth apical vesicle (arrow) after incubation at 38°C for 10 min. (E) The arrow points to a smooth transport vesicle containing a TGEV particle which is positioned close to a larger endosome-like vesicle (EN), indicating vesicle fusion. (F) TGEV particles in large intracellular vesicles after incubation at 38°C for 30 min. The arrow indicates putative continuity between viral and vesicle membranes, indicating fusion between the TGEV particle and the intracellular vesicle. Bars, 200 nm.

FIG. 2

Specific binding of TGEV to pAPN-MDCK cells. MDCK cells permanently expressing human APN (a) or pAPN-MDCK cells (b) were incubated with TGEV (solid lines) or mock incubated (dotted lines). Cellular binding of TGEV was monitored by flow cytometry after incubation with a porcine anti-TGEV serum followed by incubation with a fluorescein isothiocyanate-labelled rabbit IgG directed against porcine IgG.

FIG. 3

Colocalization of pAPN and TGEV. Localization of pAPN was carried out by immunogold labelling of pAPN-MDCK cells which were incubated with TGEV from the apical side (4°C, 1 h). The cells were then incubated at 38°C for 10 min. (A) Gold labelling demonstrating the presence of pAPN is seen just beneath the TGEV particle (arrow) and to a lesser extent in the rest of the plasma membrane. (B) Gold labelling of a TGEV-containing pit (arrow), demonstrating colocalization between TGEV and pAPN. (C) Labelling of a large intracellular vesicle. The gold particles are seen all over the membrane localized to the cisternal side beneath the TGEV particles, demonstrating the presence of pAPN in close proximity to TGEV. Bars, 200 nm.

FIG. 4

TGEV- and pAPN-free large intracellular vesicles. The cells and labelling are as in Fig. 3. The arrows indicate vesicles free of TGEV particles and pAPN labelling. The arrowhead indicates a pAPN-TGEV-containing vesicle. Bar, 200 nm.

FIG. 5

Inhibitory effect of ammonium chloride on TGEV infectivity. Virions were bound to pAPN-MDCK cells (a) or ST cells (b) at 0°C for 120 min, and unbound virions were washed away. Ammonium chloride (25 mM final concentration) was added at the indicated times (○) and was present during different 2-h periods as indicated by the horizontal bars. The ammonium chloride was washed out of the cells at the ends of these periods. (a) The cytopathic effect was quantified colorimetrically by measuring cellular crystal violet association as described in Materials and Methods. The means and standard errors from three experiments are shown. (b) The cells were overlaid with agar at 38°C and incubated for 48 h at this temperature for plaque formation. The means from two different experiments are shown.

FIG. 6

Inhibitory effect of bafilomycin A₁ (a) and ammonium chloride (b) on TGEV infectivity. Virions were bound to ST cells at 0°C for 120 min, unbound virions were washed away with ice-cold medium, and then warm medium (37°C) was added. Bafilomycin A₁ (5 μM final concentration) (a) or ammonium chloride (25 mM) (b) was added at the indicated times (minutes) after adsorption. For each time, duplicate infections were performed. At 3 h after adsorption, [³⁵S]methionine was added. After 5 h of further incubation, the cells were solubilized and the viral proteins were immunoisolated, separated by SDS-polyacrylamide gel electrophoresis, and visualized by autoradiography.