Stable expression of noncytopathic Kunjin replicons simulates both ultrastructural and biochemical characteristics observed during replication of Kunjin virus

This report focuses mainly on the characterization of a Vero cell line stab ly expressing the flavivirus Kunjin (KUN) replicon C20SDrep (C20SDrepVero). We showed by immunofluorescence and cryoimmunoelectron microscopy that uni que flavivirus-induced membrane structures, termed convoluted membranes/par acrystalline structures, were induced in the C20SDrepVero cells. These indu ced cytoplasmic foci were immunolabeled with KUN virus anti-NS3 antibodies and with antibodies to the cellular markers ERGIC53 (for the intermediate c ompartment) and protein disulfide isomerase (for the rough endoplasmic reti culum). However, in contrast to the large perinuclear inclusions observed b y immunofluorescence with anti-double-stranded (ds)RNA antibodies in KUN vi rus-infected cells, the dsRNA in C20SDrepVero cells was localized to small isolated foci scattered throughout the cytoplasm, which were coincident wit h small foci dual-labeled with the trans-Golgi specific marker GaIT. import antly persistent expression of the KUN replicons in cells did not produce c ytopathic effects, and the morphology of major host organelles (including G olgi, mitochondria, endoplasmic reticulum, and nucleus) was apparently unaf fected. The amounts of plus- and minus-sense RNA synthesis in replicon cell s were similar to those in KUN virus-infected cells until near the end of t he latent period, but subsequently increases of about 10- and fourfold, res pectively, occurred in infected cells. Virus-specified protein synthesis in C20SDrepVero cells was also about 10-fold greater than that in infected ce lls. When several KUN replicon cell lines were compared with respect to mem brane induction, the relative efficiencies increased in parallel with incre ases in viral RNA and protein synthesis, consistent with the increases obse rved during the virus infectious cycle. Based on these observations, cell l ines expressing less-efficient replicons may provide a useful tool to study early events in flavivirus RNA replication, which are difficult to assess in Virus infections. (C) 2001 Academic press.