THE CORONAVIRUS TRANSMISSIBLE GASTROENTERITIS VIRUS CAUSES INFECTION AFTER RECEPTOR-MEDIATED ENDOCYTOSIS AND ACID-DEPENDENT FUSION WITH AN INTRACELLULAR COMPARTMENT

Aminopeptidase N is a species-specific receptor for transmissible gast roenteritis virus (TGEV), which infects piglets, and for the 229E viru s, which infects humans. It is not known whether these coronaviruses a re endocytosed before fusion with a membrane of the target cell, causi ng a productive infection, or whether they fuse directly with the plas ma membrane. We have studied the interaction between TGEV and a cell l ine (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associ ated 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-cont aining population of large intracellular vesicles, morphologically com patible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surro unding plasma membrane, indicating that TGEV recruits pAPN receptors b efore endocytosis. Ammonium chloride and bafilomycin A(1) markedly inh ibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course o f the infection, i.e., about 1 h after the start of endocytosis. Toget her our results point to an acid intracellular compartment as the site of fusion for TGEV.