J. gen. Virol. (1982), 58, 47-61. Printed in Great Britain Key words: inhibition of virus entry/chtoroquine/amantadine/SFV 47 Inhibition of Semliki Forest Virus Penetration by Lysosomotropic Weak Bases By ARI HELENIUS,*t MARK MARSHt AND JUDY WHITEr European Molecular Biology Laboratory, Postfach 102209, 6900 Heidelberg, Federal Republic of Germany (Accepted 27 July 1981) SUMMARY The effect of five lysosomotropic weak bases (chloroquine, amantadine, tributylamine, methylamine and NH4CI) on Semliki Forest virus (SFV) infection has been studied in BHK-21 cells. When present at concentrations equal to or greater than 0-1, 0.5, 2, 15 and 15 mM respectively, the agents inhibited SFV infection by more than 90%. The effect was reversible and involved a process occurring within the first 60 min of virus-cell contact. The agents did not have a direct virucidal effect nor did they affect virus binding to the cells, receptor-mediated endocytosis of prebound virus, intracellular distribution of virus after endocytosis, or the low pH-induced membrane fusion activity of the virus spike glycoproteins. The step blocked by chloroquine and NH4CI occurred intracellularly and was identified as the release of the virus nucleocapsid into the cytoplasm or the uncoating process. On the basis of these results, our previous studies on SFV entry, and the known effects of lipophilic amines on lysosomes, we conclude that the agents affect entry by a common mechanism: they prevent the transfer of the virus nucleocapsid into the cytoplasm by increasing the lysosomal pH above the critical value needed to trigger a low pH-dependent fusion reaction between the membranes of the tysosome and the virus. INTRODUCTION Semliki Forest virus (SFV), an alphavirus, infects cells by an endocytotic route. The viruses are internalized by receptor-mediated endocytosis in coated vesicles and delivered into intracellular vacuoles (Helenius et al., 1980a, b; Marsh & Helenius, 1980). Our results suggest that the virus genome penetrates into the cytoplasm by fusion between the virus membrane and the limiting membrane of the secondary lysosome to which the viruses are directed (Helenius et al., 1980 a, b; White & Helenius, 1980). The fusion reaction is probably triggered by the low pH in the lysosomes (Helenius et al., 1980a, b; White & Helenius, 1980; White et al., 1980). Experiments using a lysosomotropic weak base, chloroquine, indicated that lysosomes are important in the endocytotic infection route observed at neutral pH (Helenius et al., 1980a, b; Talbot & Vance, 1980). In addition, infection could be induced directly through the plasma membrane, but only when the pH of the medium is adjusted to 6 or below. In this case the membrane of the virus fuses with the plasma membrane and the nucleocapsid is introduced into the cytoplasm (White et al., 1980). In this paper we have studied the effect of five lysosomotropic weak bases: chloroquine, t Present address: Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, U.S.A. 0022 1317/82/0000-4648 $02.00 © 1982 SGM