[Show abstract][Hide abstract] ABSTRACT: Site-directed mutagenesis of specific amino acids within a conserved amino-terminal region (H2) and a conserved carboxyl-terminal region (H10/A4) of the fusion protein G of vesicular stomatitis virus have previously identified these two segments as an internal fusion peptide and a region influencing low-pH induced conformational change, respectively. Here, we combined a number of the substitution mutants in the H2 and H10/A4 regions to produce a series of double-site mutants and determined the effect of these mutations on membrane fusion activity at acid pH and on pH-dependent conformational change. The results show that most of the double-site mutants have decreased cell-cell fusion activity and that the effects appeared to be additive in terms of inhibition of fusion, except for one mutant, which appeared to be a revertant. The double-site mutants also had pH optima for fusion that were lower than those observed with wild-type G but same as the pH optima for the parent fusion peptide (H2) mutants. The results suggest that although the H2 and H10/A4 sites may affect membrane fusion independently, a possible interaction between these two sites cannot be ruled out.
[Show abstract][Hide abstract] ABSTRACT: The envelope glycoprotein G of vesicular stomatitis virus induces membrane fusion at acidic pH. A highly conserved amino terminal region spanning residues 123 to 137 has previously been identified as an internal fusion domain. Here we have substituted specific amino acids within a carboxy terminal region, conserved in five vesiculoviruses encompassing residues 395 to 418, and studied the effect of these mutations on membrane fusion at acid pH and pH-dependent conformational change. Substitution of conserved Gly 395, Gly 404, Gly 406, Asp 409, and Asp 411 with Glu, Ala, Ala, Asn, and Asn, respectively, decreased the cell-cell fusion efficiency, as well as reduced the pH threshold of membrane fusion. Mutation of Gly 404 and Asp 409 to Lys and Ala, respectively, abolished the fusion activity. Mutant Gly 404 Lys also showed markedly altered resistance to trypsin digestion at acidic pH. These results suggest that the region between amino acids 395 to 418 is important for the fusogenic activity of the G protein. The possible role of this domain in conformational changes involved in fusion activity of VSV G is also discussed.