Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus assembly

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؇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؇C) dramatically decreased E1 cleavage and permitted assembly of morphologically normal virus particles. Pulse-labeling studies showed that the critical period for 28؇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؇C. The altered-assembly phenotype of the G91D and G91A mutants correlated with decreased stability of the E1-E2 dimer.

A Conserved Histidine in the ij Loop of the Semliki Forest Virus E1 Protein Plays an Important Role in Membrane Fusion

“…2A). H230A-infected cells released only a soluble truncated form of E1 when incubated at 37°C, similar to several other E1 mutants that are temperature sensitive for virus assembly (6,10,33). In contrast, radiolabeled H230A virus particles were efficiently released when cells were incubated at 28°C ( Fig.…”

“…Virus assembly assay. Pulse-chase experiments to evaluate virus assembly were performed essentially as described previously (10). Briefly, BHK cells were electroporated with either wt-ic or H230A-ic RNA, plated in 35-mm diameter dishes for 2 h at 37°C, and further incubated at 37 or 28°C for 4 h or overnight, respectively.…”

“…To prepare 35 Slabeled virus, BHK-21 cells were infected by electroporation with RNA, plated at 37°C for 6 h in complete BHK medium, and then radiolabeled at 28°C overnight in methionine-and cysteine-deficient MEM containing 100 Ci of [ 35 S]-methionine and cysteine per ml (PROMIX; Amersham LifeScience) as previously described (10). The virus was purified by banding on a Pfefferkorn gradient (26).…”

“…1A). The infectivity assay was also performed at 28°C, since several mutants with an assembly defect at 37°C have been shown to assemble more efficiently at 28°C (6,10). Although the spread of infection was slower than at 37°C, the wt virus showed secondary infection at 28°C, while the H230A mutant again showed no secondary infection (Fig.…”

Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus.

“…To prepare [35S]methionine and cysteine-labeled virus, BHK-21 cells were infected by electroporation with RNA, plated at 37°C for 6 h in complete BHK medium (DMEM containing 5% FCS, 100 U penicillin and 100 ~g streptomycin/ml and 10% tryptose phosphate broth), and then radiolabeled overnight in methione and cysteine deficient MEM at 28°C, all as previously described (6). Virus was then purified either by pelleting through a 2.5-ml 25% (wt/wt) sucrose cushion as previously described (6) or by banding on a Pfefferkorn gradient (21). Sucrose cushionpurified virus was used throughout except as indicated in individual experiments.…”