ACTIVE VACUOLAR H(-INFECTION OF BHK-21-CELLS()ATPASE IS REQUIRED FOR BOTH ENDOCYTIC AND EXOCYTIC PROCESSES DURING VIRAL)

Bafilomycin A(1) (Baf), a specific inhibitor of the vacuolar-type prot on pump, inhibited the entry of Semliki Forest virus and vesicular sto matitis virus into BHK-21 cells. The inhibition occurred at concentrat ions that dissipated intracellular acidic compartments. Viral infectio n 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 n eeded for virus entry. When the cells were exposed to 100 nM Eaf after virus entry, viral glycoprotein synthesis continued normally. The vir al glycoproteins acquired resistance to endoglycosidase H, indicating arrival in the medial Golgi apparatus. However, maturation processes o ccurring in the trans-Golgi compartment were inhibited, and the amount s of viral glycoproteins appearing at the cell. surface were reduced. Double immunofluorescence studies showed that in the presence of Baf t he viral glycoproteins were found in and around mannosidase II-positiv e Golgi structures. To analyze whether Baf blocked transport from the trans-Golgi compartment to the cell surface, the viral glycoproteins w ere allowed to accumulate in the trans-Golgi network by utilizing a 20 degrees C block. Subsequent incubation at 37 degrees C in the presenc e of Baf did not inhibit the terminal maturation processes or transpor t to the cell surface, suggesting that the block was before the trans- Golgi network. These results indicate that an active vacuolar-type pro ton pump in the Golgi apparatus is essential for protein transport in BHK-21 cells.