Nascent flavivirus RNA colocalized in situ with double-stranded RNA in stable replication complexes

Abstract

Incorporation of bromouridine (BrU) into viral RNA in Kunjin virus- infected Vero cells treated with actinomycin D was monitored in situ by immunofluorescence using antibodies reactive with Br-RNA. The results showed unequivocally that nascent viral RNA was located focally in the same subcellular site as dsRNA, the putative template for flavivirus RNA synthesis. When cells were labeled with BrU for 15 min, the estimated cycle period for RNA synthesis, the nascent Br-RNA was not digested in permeabilized cells by RNase A under high-salt conditions, in accord with our original model of flavivirus RNA synthesis (Chu, P. W. G., and Westaway, E. G., Virology 140, 68-79, 1985). The model assumes t..