Effects of monensin on the processing and intracellular transport of influenza virus haemagglutinin in infected MDCK cells

Abstract

The role of the Golgi complex in the intracellular transport of influenza virus haemagglutinin in infected MDCK cell monolayers has been investigated using monensin, a carboxylic ionophore known to disrupt the functioning of this organelle in other cell types. In untreated cells metabolically labelled 5 h post-infection with [35S]methionine haemagglutinin was first seen in core glycosylated form, which was sensitive to the enzyme endo-beta-N-acetylglucosaminidase H (endo H). After approximately 20 min this form was converted into a terminally glycosylated, endo H-resistant form. In the presence of monensin core glycosylation of haemagglutinin was not affected, but terminal glycosylation was interrupted. Two new forms of haemagglutinin were observed, both of which were smaller than the core glycosylated form. Of these, the larger was endo H-sensitive while the smaller was endo H-resistant. These new (and uncharacterized) forms of haemagglutinin are likely to be intermediates in the normal process of terminal glycosylation, which are revealed as a result of the inhibition by monensin of the transport of haemagglutinin through the stack of Golgi cisternae. In untreated cells 85% of the pulse-labelled haemagglutinin had reached the plasma membrane after 90 min of chase, as revealed by its sensitivity to externally applied trypsin. In monensin-treated cells, on the other hand, only 55% of the haemagglutinin had reached the plasma membrane after 90 min of chase, while 94% had arrived there after 180 min of chase. At 5 h post-infection the density of envelope proteins detected at the apical surface of the monolayer by immunofluorescence microscopy was greatly reduced by monensin treatment. Budding of virions from the apical surface of the monolayer at 4 and 7 h post-infection was also reduced, and the normal Golgi complexes were replaced by distended vacuoles that appeared to contain poorly preserved virions.