Spatial and temporal organization of tick-borne encephalitis flavivirus replicated RNA in living cells

Flaviviruses are positive RNA viruses that assemble the replication complex in the cytoplasm of the infected cells. In order to get a dynamic view of the formation and distribution of flavivirus genomes in living cells we engineered a tick-borne encephalitis virus (TBEV) replicon with an array of binding sites for the phage MS2 core protein. The modified TBEV replicons were competent for RNA replication and allowed the visualization of replicated genomic RNA that accumulated in cytoplasmic structures with a distinct subcellular localization. Sites of TBEV replicated RNA accumulation were enriched in non-structural viral proteins and co-localized with the markers of the rough endoplasmic reticulum protein disulphide isomerase (PDI). In contrast no co-localization was observed with the markers CD-71 and EEA-1 for recycling vesicles, ERGIC53 for the intermediate compartment and TGN-46 for the trans-Golgi network. In human HOS cells, but not in hamster BHK21 cells, replicated TBEV RNA was found also associated with the marker Giantin for the Golgi indicating differences according to the cellular background.This study confirms and extends previous observations on the subcellular localization of flavivirus RNA and provides a useful tool to monitor the formation and distribution of flavivirus RNA genomes in living cells.