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Effects of bafilomycin A1 on Japanese encephalitis virus in C6/36 mosquito cells
1Department of Microbiology, Saitama Medical School, Japan.
Abstract:
Involvement of intracellular acidic compartments in the early phase of Japanese encephalitis (JE) virus infection of C6/36 mosquito cells was examined by bafilomycin A1, 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 microM bafilomycin A1. In synchronized infection, cell surface-bound virions were internalized immediately by heating at 31 degrees C, followed by the release of nucleocapsid into the cytosol within a short lag period. Subcellular distribution of infecting 3H-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 1.05 g/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 A1.
Insights
Japanese encephalitis (JE) virus infection involves acidic compartments in mosquito cells. Bafilomycin A1, an inhibitor, reduced viral protein production, suggesting endocytic pathway involvement.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Japanese encephalitis (JE) virus is a significant human pathogen transmitted by mosquitoes.
- Understanding the early stages of viral entry and replication is crucial for developing antiviral strategies.
- The role of intracellular acidic compartments in JE virus infection has not been fully elucidated.
Purpose of the Study:
- To investigate the involvement of intracellular acidic compartments in the early phase of JE virus infection in C6/36 mosquito cells.
- To determine the effect of vacuolar type H(+)-ATPase (V-ATPase) inhibition on JE virus replication.
Main Methods:
- Treatment of C6/36 cells with bafilomycin A1, a specific V-ATPase inhibitor.
- Analysis of viral envelope protein (E) production.
- Synchronized infection experiments with temperature shift.
- Subcellular fractionation using Percoll density gradient centrifugation.
- Analysis of viral RNA (V-RNA) distribution and RNase sensitivity.
Main Results:
- Bafilomycin A1 treatment caused a dose-dependent reduction in viral envelope protein production.
- Internalization of cell surface-bound virions was observed upon heating.
- RNase-resistant V-RNA was detected in endosome-like fractions, while RNase-sensitive V-RNA localized to ribosomes.
- JE virus infection proceeded through an endocytic pathway sensitive to bafilomycin A1.
Conclusions:
- Intracellular acidic compartments play a critical role in the early stages of JE virus infection in C6/36 cells.
- The endocytic pathway, involving V-ATPase activity, is essential for JE virus entry and/or replication.
- Bafilomycin A1 effectively inhibits JE virus infection by targeting these acidic compartments.