Monensin Inhibits Semliki Forest Virus Penetration into Culture Cells

The carboxylic ionophores monensin and nigericin, at concentrations higher than 10 and 6 muM, respectively, prevent the penetration of the Semliki Forest virus (SFV) genome into the cytosol of baby hamster kidney (BHK-21) cells and thereby inhibit viral replication. In the absence of inhibitors, the entry of SFV is known to proceed by adsorptive endocytosis in coated vesicles, followed by acid-triggered membrane fusion in intracellular vacuoles or lysosomes. The results show that binding of the virus to the cell surface, adsorptive endocytosis, and intracellular transport of viruses to the lysosomes are only marginally affected by the ionophores. No direct virucidal effect is observed, nor is the membrane fusion activity of the virus at low pH directly affected. Sequential addition of monensin and ammonium chloride (a non-related lysosomotropic inhibitor of SFV entry) indicates that both inhibitors affect the same step in the entry pathway. On the basis of these data and the known effects of carboxylic ionophores and lysosomotropic weak bases on cellular pH gradients, we conclude that monensin inhibits penetration by increasing the pH in endocytic vacuoles and lysosomes above pH 6, which is the pH threshold for the viral membrane fusion activity.