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Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus
W A Duffus1, P Levy-Mintz, M R Klimjack
1Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Abstract:
The two transmembrane spike protein subunits of Semliki Forest virus (SFV) form a heterodimeric complex in the rough endoplasmic reticulum. This complex is then transported to the plasma membrane, where spike-nucleocapsid binding and virus budding take place. By using an infectious SFV clone, we have characterized the effects of mutations within the putative fusion peptide of the E1 spike subunit on spike protein dimerization and virus assembly. These mutations were previously demonstrated to block spike protein membrane fusion activity (G91D) or cause an acid shift in the pH threshold of fusion (G91A). During infection of BHK cells at 37 degrees C, virus spike proteins containing either mutation were efficiently produced and transported to the plasma membrane, where they associated with the nucleocapsid. However, the assembly of mutant spike proteins into mature virions was severely impaired and a cleaved soluble fragment of E1 was released into the medium. In contrast, incubation of mutant-infected cells at reduced temperature (28 degrees C) dramatically decreased E1 cleavage and permitted assembly of morphologically normal virus particles. Pulse-labeling studies showed that the critical period for 28 degrees C incubation was during virus assembly, not spike protein synthesis. Thus, mutations in the putative fusion peptide of SFV confer a strong and thermoreversible budding defect. The dimerization of the E1 spike protein subunit with E2 was analyzed by using either cells infected with virus mutants or mutant virus particles assembled at 28 degrees C. The altered-assembly phenotype of the G91D and G91A mutants correlated with decreased stability of the E1-E2 dimer.
Insights
Mutations in Semliki Forest virus (SFV) E1 fusion peptide disrupt virus assembly. Lowering temperature rescues this budding defect, revealing a thermoreversible process linked to E1-E2 dimer stability.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Semliki Forest virus (SFV) spike proteins (E1/E2 heterodimer) are crucial for virus assembly and budding.
- The E1 subunit's fusion peptide is essential for membrane fusion, but its role in assembly is less understood.
Purpose of the Study:
- To investigate the impact of mutations in the E1 fusion peptide on SFV spike protein dimerization and virus assembly.
- To characterize the thermolability of assembly defects caused by these mutations.
Main Methods:
- Utilized an infectious SFV clone with mutations in the E1 fusion peptide (G91D, G91A).
- Infected BHK cells and analyzed spike protein transport, nucleocapsid association, and virion assembly at different temperatures (37°C and 28°C).
- Employed pulse-labeling studies and analyzed E1-E2 dimer stability in mutant viruses.
Main Results:
- Mutations in the E1 fusion peptide impaired virus assembly and led to E1 cleavage at 37°C.
- Incubation at 28°C significantly reduced E1 cleavage and restored the assembly of morphologically normal virus particles.
- The assembly defect was thermoreversible, with reduced E1-E2 dimer stability correlating with the G91D and G91A mutations.
Conclusions:
- Mutations in the SFV E1 fusion peptide induce a temperature-sensitive budding defect.
- This defect is reversible by lowering the incubation temperature, highlighting the critical role of E1-E2 dimer stability in virus assembly.