ULTRASTRUCTURE OF KUNJIN VIRUS-INFECTED CELLS - COLOCALIZATION OF NS1AND NS3 WITH DOUBLE-STRANDED-RNA, AND OF NS2B WITH NS3, IN VIRUS-INDUCED MEMBRANE STRUCTURES

The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy w ith monospecific antibodies, Comparisons were also made by dual immuno labelling using antibodies to double-stranded RNA (dsRNA), the putativ e template in the flavivirus replication complex, At 8 h postinfection , the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA loca ted in a perinuclear rim with extensions into the peripheral cytoplasm , By 16 h, at the end of the latent period, all patterns had changed t o some discrete perinuclear foci associated with a thick cytoplasmic r eticulum, By 24 h, this localization in perinuclear foci was more appa rent and some foci were dual labelled with antibodies to dsRNA, In imm uno-gold-labelled cryosections of infected cells at 24 h, all antibodi es were associated with clusters of induced membrane structures in the perinuclear region, Two important and novel observations were made, F irst, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 anti bodies, Second, adjacent masses of paracrystalline arrays or of convol uted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies, Paired membranes similar in appearance to the rough endoplasmic reticulum we re also labelled, but less strongly, with antibodies to the three nons tructural proteins, Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labe lled with any of the four antibodies, The collection of induced membra nes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.