Association of Virus Specific Replicative Ribonucleic Acid with Nuclear Membrane in Chick Embryo Cells Infected with Japanese Encephalitis Virus

Abstract

SUMMARYUsing high resolution electron microscopic autoradiography and velocity sedimentation, RNA synthesis was examined in chick embryo cells infected with Japanese encephalitis virus (JEV). RNA was labelled with 3H-uridine for 5 min or lO rain at I5 h after infection in the presence of actinomycin D and D-glucosamine. Microautoradiography showed significant numbers of silver grains on the nuclear membranes of 5 rain pulse-labelled thin cell sections.The RNA species in membrane fractions obtained from the nucleus and cytoplasm of the infected cells were analysed by sucrose density gradient sedimentation. Radioactive 23S replicative form RNA and 8-I2S RNA were obtained from the outer membrane fractions of the nuclear envelope. Labelled 42S virus RNA was obtained from the fractions containing large vesicle membranes and plasma membranes.These results suggest that JEV-RNA synthesis is initiated in the perinuclear region in close association with the outer membranes of the nuclear envelope.

Localization and Functions of Japanese Encephalitis Virus Nonstructural Proteins NS3 and NS5 for Viral RNA Synthesis in the Infected Cells

“…This complex may be the same as previously reported (17,21,27). It is also similar to the poliovirus-replication complex which contained RNA polymerase, viral protease, 2C protein and viral RNA on the vesicular membranes (23), and was observed by electron microscope (1).…”

Replication of Flaviviruses

“…Ribosome like particles are often observed to stud the cytoplasmic side of the vesicles within which mature flavivirions accumulate in infected cells (Matsumura et al, 1971). Autoradiographs of actinomycin-D-treated, JE-virus-infected cells labeled with [3H]uridine for 10-30 min showed an increasing accumulation of radioactivity on rough endoplasmic reticulum (Takeda et al, 1978;Lubiniecki and Henry, 1974). These results indicate that the flavivirus proteins may be synthesized on membranes at the site of viral RNA synthesis and virion maturation.…”

“…Intracellular viral RNA synthesis has been detected as early as 3 hr after infection (Takeda et al, 1978). Trent et al (1969) found that flavivirus RNA synthesis was biphasic, with a small peak of synthesis occurring at 6 hr after infection and then a steady increase in RNA synthesis beginning by 13 hr after infection.…”

“…Flavivirus in vitro polymerase activity has been detected in crude infected cellular extracts by several laboratories (Zebovitz et al, 1974;Takeda et al, 1978;Brawner et al, 1979;Cardiff et al, 1973;Qureshi and Trent, 1972;Chu and Westaway, 1985;Grun and Brinton, 1986a and b). Polymerase activity was found to be reduced by treatment with nonionic detergents (Cardiff et al, 1973), indicating an association of the active replication complexes with membranes.…”

“…Polymerase activity is associated with cytoplasmic membranes, the mitochondrial fraction, and nuclear-associated membranes. However, activity was consistently found to be highest in nuclear-associated membranes (Zebovitz et al, 1974;Takeda et al, 1978;Chu and Westaway, 1985;Grun and Brinton, 1986a). This finding is not unexpected, since the majority of viral RNA synthesis occurs in the perinuclear region of infected cells (Westaway, 1980).…”