Assembly of enveloped viruses in Madin-Darby canine kidney cells: polarized budding from single attached cells and from clusters of cells in suspension.

In confluent monolayers of the dog kidney epithelial cell line Madin-Darby canine kidney (MDCK) assembly of RNA enveloped viruses reflects the functional polarization of the cells. Thus, influenza, Sendai, and Simian virus 5 bud from the apical (free) surface, while vesicular stomatitis virions (VSV) are assembled at basolateral plasma membrane domains (Rodriguez-Boulan, E., and D.D. Sabatini, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:5071-5075). MDCK cells derived from confluent monolayers by dissociation with trypsin-EDTA and maintained as single cells in spinner medium for 12-20 h before infection, lose their characteristic structural polarity. Furthermore, when these cells were infected with influenza or VSV, virions assembled in a nonpolarized fashion over most of the cell surface. However, when dissociated MDCK cells infected in suspension were sparsely plated on collagen gels to prevent intercellular contact and the formation of junctions, the characteristic polarity of viral budding observed in confluent monolayers was again manifested; i.e., VSV budded preferentially from adherent surfaces and influenza almost exclusively from free surface regions. Similar polarization was observed in cells which became aggregated during incubation in spinner medium: influenza budded from the free surface, while VSV was produced at regions of cell-cell contact. It therefore appears that in isolated epithelial cells attachment to a substrate or to another cell is sufficient to trigger the expression of plasma membrane polarity which is manifested in the asymmetric budding of viruses.

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

The intracellular 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 degrees C, exhibits a defect in the HA that prevents its exit from the endoplasmic reticulum. Upon transfer to permissive temperature, 32 degrees 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 microM) delayed the surface appearance of HA by 2 h, but not the transport to the Golgi apparatus. Incubation at 20 degrees C retarded the migration to the Golgi apparatus by approximately 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.