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-GI) or the heterologous glycoprotein gene from the New Jersey serotype (VSIV-GNJ). The virus expressing the GNJ gene was more pathogenic than the parental VSIV-GI virus in swine, a natural host (26). For the present study, we investigated the biological differences between the GI and GNJ proteins that may be related to the differences in pathogenesis between VSIV-GI and VSIV-GNJ. We show that the capacities of viruses with either the GNJ or GI glycoprotein to infect cultured cells differ depending on the pH. VSIV-GNJ could infect cells at acidic pHs, while the infectivity of VSIV-GI was severely reduced. VSIV-GNJ infection was also more sensitive to inhibition by ammonium chloride, indicating that the GNJ protein had a lower pH threshold for membrane fusion. We applied selective pressure to VSIV-GI 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-GI variant (VSIV-6.8) that was similar to VSIV-GNJ 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-GI 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.
Martinez
Isidoro
I
Department of Microbiology, University of Alabama School of Medicine, BBRB Box 17, Room 366, 845 19th St. South, Birmingham, AL 35294, USA.
Wertz
Gail W
GW
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D012641:Selection, Genetic; D012703:Serotyping; D014709:Vero Cells; D014721:Vesicular stomatitis Indiana virus / Q000145:classification / Q000235:genetics* / Q000502:physiology*; D018116:Vesiculovirus; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010948:Viral Plaque Assay
D000596:Amino Acids; D006023:Glycoproteins; D014759:Viral Envelope Proteins
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