Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

Abstract

Temperature-sensitive (ts) mutants of Semliki Forest virus (SFV) and Sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chick embryo fibroblasts. When antisera against purified glycoproteins and 125I-labeled protein A from Staphylococcus arueus were used, only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN ts-10 infected cells incubated at the restrictive temperature (39.degree. C). When the mutant-infected cells were shifted to the permissive temperature (28.degree. C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20-80 min after the shift. Monensin (10 .mu.M) and carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP; 10-20 .mu.M) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 .mu.g/ml) inhibited the transport by .apprx. 50%; cytochalasin B (1 .mu.g/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39.degree. C, the virus membrane glycoproteins were located at the endoplasmic reticulum; after shift to 28.degree. C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins migrated to the Golgi apparatus, although transport to the cell surface did not occur. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period; with monensin there was a delay of .apprx. 10 min. These results suggest a similar intracellular pathway in the maturation of plasma membrane and secretory glycoproteins.