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

Incorporation of bromouridine (BrU) into viral RNA in Kunjin virus-infected Vero cells treated with actinomycin D was monitored in situ by immunofluor escence using antibodies reactive with Br-RNA. The results showed unequivoc ally that nascent viral RNA was located focally in the same subcellular sit e as dsRNA, the putative template for flavivirus RNA synthesis. When cells were labeled with BrU for 15 min, the estimated cycle period for RNA synthe sis, 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 that there is on average only one nascent strand per t emplate, which remains bound until displaced during the next cycle of RNA s ynthesis. The replicase complex located by BrU incorporation in the identif ied foci was stable, remaining active in incorporating BrU or [P-32]orthoph osphate in viral RNA after complete inhibition of protein synthesis in cycl oheximide-treated cells. These results are in accord with our proposal that dsRNA detected in foci previously located by immunofluorescence or by immu nogold labeling of induced vesicle packets is functioning as the true repli cative intermediate (Westaway at al., J. Virol. 71, 6650-6661, 1997; Macken zie el ai., Virology 245, 203-215, 1998). Implications are that the replica se complex is able to recycle in the same membrane site in the absence of c ontinuing protein synthesis and that possibly apart from uncleaved NS3-NS4A , it has no requirement for a polyprotein precursor late in infection. (C) 1999 Academic Press.