Active vacuolar H+ATPase is required for both endocytic and exocytic processes during viral infection of BHK-21 cells.

Bafilomycin A1 (Baf), a specific inhibitor of the vacuolar-type proton pump, inhibited the entry of Semliki Forest virus and vesicular stomatitis virus into BHK-21 cells. The inhibition occurred at concentrations that dissipated intracellular acidic compartments. Viral infection was totally inhibited by 30 nM Baf while endocytosis of the virus or fluorescein isothiocyanate-dextran was not affected. Thus, a vacuolar-type proton pump was responsible for acidification of the endosomes needed for virus entry. When the cells were exposed to 100 nM Baf after virus entry, viral glycoprotein synthesis continued normally. The viral glycoproteins acquired resistance to endoglycosidase H, indicating arrival in the medial Golgi apparatus. However, maturation processes occurring in the trans-Golgi compartment were inhibited, and the amounts of viral glycoproteins appearing at the cell surface were reduced. Double immunofluorescence studies showed that in the presence of Baf the viral glycoproteins were found in and around mannosidase II-positive Golgi structures. To analyze whether Baf blocked transport from the trans-Golgi compartment to the cell surface, the viral glycoproteins were allowed to accumulate in the trans-Golgi network by utilizing a 20 degrees C block. Subsequent incubation at 37 degrees C in the presence of Baf did not inhibit the terminal maturation processes or transport to the cell surface, suggesting that the block was before the trans-Golgi network. These results indicate that an active vacuolar-type proton pump in the Golgi apparatus is essential for protein transport in BHK-21 cells.