ATTENUATION OF RECOMBINANT VESICULAR STOMATITIS VIRUSES ENCODING MUTANT GLYCOPROTEINS DEMONSTRATE A CRITICAL ROLE FOR MAINTAINING A HIGH PHTHRESHOLD FOR MEMBRANE-FUSION IN VIRAL FITNESS

A plasmid-based recovery system was used to generate four unique vesic ular stomatitis virus (VSV) mutants that encode glycoproteins (G prote ins) with single or double amino acid substitutions in two conserved a cidic residues adjacent to the putative G protein fusion domain. Previ ously we demonstrated that three of the mutant G proteins (D137-L, E13 9-L, and DE-SS) have slightly reduced pH thresholds for membrane fusio n activity. In this report we show that even though the viruses encodi ng D137-L, E139-L, and DE-SS were recovered with high efficiency, thes e mutants were attenuated for growth in cell culture. Plaque formation was significantly delayed with these mutants and the plaques were sma ller and more diffuse than those produced by wild-type VSV. In additio n, cells infected with these mutants produced approximately 5- to 10-f old less infectious virus than cells infected with a similarly recover ed VSV encoding the wild-type G protein. Using R18-labeled virus we fo und that the mutant G proteins had approximately 50% of the fusion act ivity of wild-type G at pH 6.3 and only 75% activity at pH 5.8. We als o show that the mutant viruses were more sensitive to chloroquine inhi bition of infection than either wild-type VSV or the mutant E139-T, wh ich has a fusion phenotype similar to wild-type G protein. Reduced fus ion activity and attenuation of infectivity was not due to differences in the amount of G protein incorporated into virions, nor to differen ces in the amount of virus binding to cells at physiological pH. Altho ugh infectivity was assayed at neutral pH, we observed an increase in virus binding with both mutant and wild-type virions as the pH was low ered, and the increase in binding occurred near the pH threshold for m embrane fusion activity. From these data we propose a model in which V SV entry involves an increase in virus binding to the inner leaflet of the endosomal membrane during endosome acidification. Concomitant wit h this higher affinity binding, G protein becomes primed to initiate f usion of the viral envelope with the endosomal membrane. Viruses with mutations that delay the onset of increased binding and fusion lag beh ind wild-type VSV in their ability to initiate a productive infection, potentially because the location within the cytoplasm where these vir uses ultimately fuse is not optimal for either virus uncoating or repl ication of the viral genome. (C) 1998 Academic Press.