REQUIREMENT FOR VACUOLAR PROTON-ATPASE ACTIVITY DURING ENTRY OF INFLUENZA-VIRUS INTO CELLS

The role that endosomal acidification plays during influenza virus ent ry into MDCK cells has been analyzed by using the macrolide antibiotic s bafilomycin A1 and concanamycin A as selective inhibitors of vacuola r proton-ATPase (v-[H+]ATPase), the enzyme responsible for the acidifi cation of endosomes. Bafilomycin A1 and concanamycin A, present at the low concentrations of 5 x 10(-7) and 5 x 10(-9) M, respectively, prev ented the entry of influenza virus into cells when added during the fi rst minutes of infection. Attachment of virion particles to the cell s urface was not the target for the action of bafilomycin A1. N,N'-Dicyc lohexylcarbodiimide, a nonspecific inhibitor of proton-ATPases, also b locked virus entry, whereas elaiophylin, an inhibitor of the plasma-pr oton ATPase, had no effect. The inhibitory actions of bafilomycin A1 a nd concanamycin A were tested in culture medium at different pHs. Both antibiotics powerfully prevented influenza virus infection when the v irus was added under low-pH conditions. This inhibition was reduced if the virus was bound to cells at 4 degrees C prior to the addition of warm low-pH medium. Moreover, incubation of cells at acidic pH potentl y blocked influenza virus infection, even in the absence of antibiotic s. These results indicate that a pH gradient, rather than low pH, is n ecessary for efficient entry of influenza virus into cells.