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Membrane fusion mutants of Semliki Forest virus
Abstract:
Previous reports have indicated that the entry of Semliki Forest virus (SFV) into cells depends on a membrane fusion reaction catalyzed by the viral spike glycoproteins and triggered by the low pH prevailing in the endosomal compartment. In this study the in vitro pH-dependent fusion of SFV with nuclease-filled liposomes has been used to select for a new class of virus mutants that have a pH-conditional 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 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, moreover, able to penetrate and infect baby hamster kidney-21 cells at 20 degrees C, indicating that the endosomes have a pH below 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.
Insights
New Semliki Forest virus (SFV) mutants exhibit pH-conditional fusion defects, altering viral entry mechanisms. These SFV mutants serve as valuable biological probes for studying the endocytic pathway and its pH dynamics.
Area of Science:
- Virology
- Cell Biology
- Membrane Fusion
Background:
- Semliki Forest virus (SFV) entry into host cells is known to involve membrane fusion.
- This fusion process is typically triggered by the low pH environment within the endosome.
- Viral spike glycoproteins catalyze this pH-dependent fusion reaction.
Purpose of the Study:
- To investigate the role of pH in SFV entry by generating and characterizing pH-conditional fusion mutants.
- To utilize these mutants as biological tools for probing the endocytic pathway.
Main Methods:
- In vitro pH-dependent fusion assays using SFV and nuclease-filled liposomes.
- Selection of SFV mutants with altered fusion pH thresholds.
- Assays included polykaryon formation, liposome fusion, and plasma membrane fusion.
- Infection studies in baby hamster kidney-21 cells at different temperatures.
Main Results:
- A novel class of SFV mutants with a higher fusion pH threshold (5.5) compared to wild-type (6.2) was identified.
- These mutants showed normal infectivity under standard conditions but increased sensitivity to lysosomotropic agents.
- Mutant SFV demonstrated efficient infection at 20°C, suggesting endosomal pH is below 5.5.
- The findings confirm pH-triggered fusion as critical for SFV entry.
Conclusions:
- The study confirms the essential role of pH-triggered membrane fusion in Semliki Forest virus entry.
- Acidic endosomal vacuoles are central to this fusion mechanism.
- Lysosomotropic weak bases inhibit SFV by increasing endosomal pH.
- SFV mutants are effective biological pH probes for the endocytic pathway.