Mutagenesis of the putative fusion domain of the Semliki Forest virus spike protein
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Semliki Forest virus (SFV), an enveloped alphavirus, infects cells by an acid-dependent membrane fusion reaction triggered by treatment at pH 6.5 or below. Fusion is mediated by the viral spike protein, a heterotrimer of two transmembrane subunits, E1 and E2, and a peripheral protein, E3. A 23 amino acid long hydrophobic domain located 75 residues from the N-terminus of E1 is highly conserved among all of the alphaviruses. Surprisingly, a homologous region is also found on the VP5 outer capsid protein of the non-enveloped rotaviruses.;We used site directed mutagenesis to test the role of this region in membrane fusion. Wild type and mutant spiked proteins were expressed in COS cells using an SV40 vector, and low pH-dependent cell-cell fusion was visualized and quantitated by immunofluorescence and nuclear staining. An antibody-protein A assay was used to determine the cell surface expression of mutant spike proteins and to normalize expression to wild type levels. Four classes of mutants were characterized in our studies. More than 80% cell-cell fusion was observed after pH 6.2 treatment of spike proteins containing a Lys to Gln change at position 79 or a Met to Leu change at position 88. These mutants were indistinguishable from wild type with respect to pH threshold and efficiency of fusion. Substitution of Asp for Pro86, or Pro for Gly91, or deleting ten amino acids of the conserved core of the hydrophobic domain caused retention of the E1 subunit in the ER. Subsequent cell-cell fusion assays with these mutants showed no fusion activity, confirming that the fusogenic E1 subunit was not present at the cell surface. The substitution of an Ala for Asp75, Gly83 or Gly91 markedly shifted the pH threshold to a more acidic range. The overall fusion efficiency of the mutants at positions 75 and 91 was also reduced by 30%. Lastly, a mutant in which Gly91 was changed to Asp completely blocked cell-cell fusion at all acidic pH values (pH 6.5-4.7), without affecting spike protein assembly or transport.;Our results suggest that the conserved hydrophobic domain of the alphavirus E1 protein is intimately involved in membrane fusion activity. Since a mutation of Gly91 could completely block activity, one may hypothesize that this residue plays a key role in virus membrane fusion.