Attenuation of recombinant vesicular stomatitis viruses encoding mutant glycoproteins demonstrate a critical role for maintaining a high pH threshold for membrane fusion in viral fitness.

摘要

When D137-L, E139-L and DE-SS mutations were introduced into the G gene of a full-length cDNA clone of vesicular stomatitis virus (VSV), the mutants were recovered with high efficiency. However, virions containing D137-L, E139-L and DE-SS were attenuated for growth in cell culture, and plaques of the 3 mutant viruses were not visible until nearly 48 h post-infection and had a diffuse appearance. Supernatant collected after 24 h from cells infected with the mutants contained 5- to 10-fold lower titres than wild-type VSV. In contrast, the growth characteristics of E139-T, which was also recovered, were very similar to those of the wild-type. When radiolabelled virus was incubated with BHK cells in buffered media, D137-L and E139-L showed slightly reduced binding activity at pH 6.5. In an octadecylrhodamine assay, D137-L, E139-L and DE-SS exhibited reduced fusion activity compared to the wild-type at pH 5.8 and above. D137-L and DE-SS were more sensitive to chloroquine inhibition of [3H]uridine incorporation than wild-type VSV. A model for VSV entry, based on the results of these experiments, is proposed.