Expression of alphavirus glycoproteins on the surface of infected cells leads to cell fusion after exposure to acidic pH. Two strains of Sindbis virus, AR339 (SV) and neuroadapted Sindbis virus (NSV), which differ in virulence for weanling mice, were found to differ in pH-dependent fusion. BHK-21 cells infected with SV fused maximally after shifting to pH 5.4, whereas cells infected with NSV required a lower pH, pH 4.8, for maximal fusion. No difference was noted in the optimal pH for agglutination of goose erythrocytes (5.75 for both viruses). To determine the molecular basis for the difference in fusion a series of recombinant viruses was constructed using a cDNA clone of Sindbis virus from which infectious RNA can be transcribed in vitro. Cells infected with a recombinant virus that had the SV E1 and NSV E2 genes had a fusion response curve as a function of pH like SV, while cells infected with recombinant virus with the NSV E1 and SV E2 genes fused like NSV. The E1 glycoproteins of SV and NSV differ at two positions: Val-72 in SV is Ala in NSV (a change near the putative fusion site), and Gly-313 in SV is Asp in NSV. Recombinant viruses which had Val-72 (SV) and Asp-313 (NSV) or Ala-72 (NSV) and Gly-313 (SV) had a lowered pH of fusion like NSV suggesting that both positions participate in determining some aspect of the conformational change in the E1-E2 heterodimer associated with pH-dependent fusion.
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