Intracellular transport of influenza virus hemagglutinin to the apical surface of Madin-Darby canine kidney cells

Abstract

The intracellular transport pathway followed by the influenza virus hemagglutinin (HA) to the apical surface of Madin-Darby canine kidney cells was studied by radioimmunoassay, immunofluorescence, and immunoelectron microscopy. To synchronize the migration, we used a temperature-sensitive mutant of influenza WSN, ts61, which, at the nonpermissive temperature, 39.5° C, exhibits a defect in the HA that prevents its exit from the endoplasmic reticulum. Upon transfer to permissive temperature, 32°C, the HA appeared in the Golgi apparatus after 10 min, and on the apical surface after 30-40 min. In the presence of cycloheximide, the expression was not inhibited, indicating that the ts defect is reversible; a wave of HA migrated to the cell surface, where it accumulated with a half time of 60 min. After passage through the Golgi apparatus the HA was detected in a population of smooth vesicles, about twice the size of coated vesicles, located in the apical half of the cytoplasm. These HA-containing vesicles did not react with anti-clathrin antibodies. Monensin (10 μM) delayed the surface appearance of HA by 2 h, but not the transport to the Golgi apparatus. Incubation at 20°C retarded the migration to the Golgi apparatus by ~30 min and blocked the surface appearance by acting at a late stage in the intracellular pathway, presumably at the level of the post-Golgi vesicles. The initial appearance of HA on the apical surface was in the center; no preference was observed for the tight-junctional regions.