Japanese encephalitis virus RNA synthesis in vivo and in vitro

概要

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In order to investigate the mechanism of Japanese encephalitis virus (JEV) RNA replication, viral RNA syntheses in vivo and in vitro were analyzed. First, an appearance of viral specific RNA in JEV-infected cells was examined by northern blot analysis. Minus RNA-probe recognized a negative strand of JEV-specific RNA synthesized in JEV-infected cells as early as 6 hrs post infection (p.i.). Full length genomic 42S positive RNAs were detected in the cells at 12 hrs p.i. Relative amounts of the 42S positive RNA was much larger in the membrane fraction than the supernatant fraction of JEV-infected cells. To investigate the mechanism of RNA synthesis, it is important to establish the in vitro system of RNA synthesis. It was found that JEV specific positive RNAs were efficiently synthesized in vitro in the crude membrane and nuclear fractions prepared from the JEV-infected cells. The analyses by SDS-PAGE and immunofluorescence assay indicated that nonstructural proteins NS3 and NS5, considered to be RNA helicase, protease and RNA polymerase, respectively, were membrane associated proteins, even though they were hydrophilic proteins. Maybe other NS proteins, including NS4a and 4b, are responsible to the membrane association, because of the hydrophobicity. The data that monospecific antisera against NS3 and NS5 inhibited in vitro RNA synthesis indicate that those proteins strongly contributed for viral RNA synthesis in the JEV-infected cells.