Biological differences between vesicular stomatitis virus Indiana and New Jersey serotype glycoproteins: Identification of amino acid residues modulating pH-dependent infectivity

We previously generated recombinant vesicular stomatitis viruses (VSV) based on the Indiana serotype genome which contained either the homologous glycoprotein gene from the Indiana serotype (VSIV-G(I)) or the heterologous glycoprotein gene from the New Jersey serotype (VSIV-G(NJ)). The virus expressing the G(NJ) gene was more pathogenic than the parental VSIV-G(I) virus in swine, a natural host (26). For the present study, we investigated the biological differences between the G(I) and G(NJ) proteins that may be related to the differences in pathogenesis between VSIV-G(I) and VSIV-G(NJ). We show that the capacities of viruses with either the G(NJ) or G(I) glycoprotein to infect cultured cells differ depending on the pH. VSIV-G(NJ) could infect cells at acidic pHs, while the infectivity of VSIV-G(I) was severely reduced. VSIV-G(NJ) infection was also more sensitive to inhibition by ammonium chloride, indicating that the G(NJ) protein had a lower pH threshold for membrane fusion. We applied selective pressure to VSIV-G(I) by growing it at successively lower pH values and isolated variant viruses in which we identified amino acid changes that conferred low-pH-resistant infectivity. Repeated passage in cell culture at pH 6.8 resulted in the selection of a VSIV-G(I) variant (VSIV-6.8) that was similar to VSIV-G(NJ) regarding its pH- and ammonium chloride-dependent infectivity. Sequence analysis of VSIV-6.8 revealed that it had a single amino acid substitution in the amino-terminal region of the glycoprotein (F18L). This alteration was shown to be responsible for the observed phenotype by site-directed mutagenesis of a VSIV-G(I) full-length cDNA and analysis of the recovered engineered virus. A further adaptation of VSIV-6.8 to pHs 6.6 and 6.4 resulted in additional amino acid substitutions in areas of the glycoprotein that were not previously implicated in attachment or fusion.