MUTATIONS IN THE PUTATIVE FUSION PEPTIDE OF SEMLIKI FOREST VIRUS AFFECT SPIKE PROTEIN OLIGOMERIZATION AND VIRUS ASSEMBLY

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-n ucleocapsid binding and virus budding take place. By using an infectio us SFV clone, we have characterized the effects of mutations within th e putative fusion peptide of the E1 spike subunit on spike protein dim erization and virus assembly. These mutations were previously demonstr ated to block spike protein membrane fusion activity (G91D) or cause a n acid shift in the pH threshold of fusion (G91A). During infection of BHK cells at 37 degrees C, virus spike proteins containing either mut ation were efficiently produced and transported to the plasma membrane , where they associated with the nucleocapsid. However, the assembly o f mutant spike proteins into mature virions was severely impaired and a cleaved soluble fragment of E1 was released into the medium. In cont rast, incubation of mutant-infected cells at reduced temperature (28 d egrees 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 putativ e fusion peptide of SFV confer a strong and thermoreversible budding d efect. The dimerization of the E1 spike protein subunit with E2 was an alyzed by using either cells infected with virus mutants or mutant vir us particles assembled at 28 degrees C. The altered-assembly phenotype of the G91D and G91A mutants correlated with decreased stability of t he E1-E2 dimer.