Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts.

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.

Reduced extracellular pH reversibly inhibits oligomerization, intracellular transport, and processing of the influenza hemagglutinin in infected Madin-Darby canine kidney cells.

Incubation of Madin-Darby canine kidney cells infected with influenza virus in medium of pH 5.8-6.0 blocks transport of newly synthesized hemagglutinin and processing of the hemagglutinin oligosaccharides to a form resistant to endo H digestion. Upon restoration of the culture medium to pH 7.4, arrested hemagglutinin is processed and then appears on the cell surface, indicating that exclusively transport and not oligosaccharide processing is inhibited. Based upon kinetic data and localization of blocked hemagglutinin by immunofluorescence, the point of inhibition appears to be a discrete step in transport located in a pre-Golgi compartment. This conclusion is supported by the observation that trimerization of hemagglutinin, which is believed to occur in the endoplasmic reticulum, is also inhibited by acidic medium.