Membrane fusion mutants of Semliki Forest virus.

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Abstract
The in vitro pH-dependent fusion of Semliki-Forest virus (SFV) with nuclease-filled liposomes was used to select for a new class of virus mutants that have a pH-conditional fusion defect. The mutants obtained had a threshold for fusion of pH 5.5 as compared with the wild-type threshold of 6.2, when assayed by polykaryon formation, fusion with liposomes or fusion at the plasma membrane. They were fully capable of infecting chicken embryo fibroblast cells under standard infection conditions but were more sensitive to lysosomotropic agents that increase the pH in acidic vacuoles of the endocytic pathway. The mutants were able to penetrate and infect baby hamster kidney (BHK-21) cells at 20.degree. C, indicating that the endosomes have a pH < 5.5. The results confirm the involvement of pH-triggered fusion in SFV entry, emphasize the central role played by acidic endosomal vacuoles in this reaction, shed further light on the mechanism of SFV inhibition by lysosomotropic weak bases and demonstrate the usefulness of mutant viruses as biological pH probes of the endocytic pathway.

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