Effects of bafilomycin Aff1 on Japanese encephalitis virus in C6/36 mosquito cells

Summary

Involvement of intracellular acidic compartments in the early phase of Japanese encephalitis (JE) virus infection of C6/36 mosquito cells was examined by bafilomycin Aff1, a specific inhibitor of vacuolar type H+-ATPase (V-ATPase). Dose dependent reduction of viral envelope protein (E) produced into the infected culture fluid was observed by pretreating the cells with 0.25 to 1.0 μM bafilomycin Al. In synchronized infection, cell surface-bound virions were internalized immediately by heating at 31 °C, followed by the release of nucleocapsid into the cytosol within a short lag period. Subcellular distribution of infecting ²H-uridine-labeled viral RNA (V-RNA) and its RNase sensitivity were analyzed by fractionation in Percoll density gradient centrifugation.

At a 10 min chasing period, an RNase resistant V-RNA peak was found in fractions with a mean density of 1.05g/ml corresponding to the endosome, while an RNase sensitive V-RNA peak was detected at density range of 1.052–1.054 g/ml corresponding to the ribosome in C6/36 cell homogenate. The results indicate that JE virus infection in C6/36 cells proceeded through the endocytic pathway involving intracellular acidic compartments which was affected by bafilomycin Aff1.