Figure 1

Schematic representation of WNV replicon constructs and packaging of WNV reporter replicon particles (RRPs). (a) The DNA based WNV replicon is under control of the CMV promoter. The replicon genome lacks the major coding sequence of the structural protein, C-prM-E, and the corresponding sequence was replaced with a GFP coding sequence following the FMDV 2A coding sequence. The replicon RNA with an authentic 5′ terminus is ensured by placing a hammerhead ribozyme sequence (HRr) before the first 5′ UTR nucleotide. The authentic 3′ terminus is ensured by the addition of a hepatitis delta virus ribozyme sequence (HDVr) after the last 3′ UTR nucleotide. To complement the structural proteins, a capsid protein expressing plasmid was constructed. The BWNV-ME cell line stably expressing the prM-E protein was generated via transfecting BHK-21 cells with the pCAG-WNV-ME plasmid. The BWNV-CME cell line stably expressing the C-prM-E protein was generated by transfecting BHK-21 cells with the pCAG-WNV-CME plasmid. (b) WNV RRPs are packaged in two ways. First, the pCAG-WNV-C and replicon plasmids were transfected into BWNV-ME cells. The pCAG-WNV-C plasmid expressed protein C. The cell itself expressed prM and E protein. The replicon plasmid was transcribed by the CMV promoter into the replicon RNA expressing GFP and the non-structural replicase protein. The replicon RNA amplifies itself once again and three structural proteins package the replicon RNA into WNV RRPs which are secreted into the culture medium. The second way is to transfect BWNV-CME cells with only the replicon plasmid. The BWNV-CME cells express the C-prM-E polyprotein which is cleaved into the C, prM and E proteins by replicon RNA-encoded non-structural protease and endogenous cellular signal peptidase (SP). Then three structural proteins package the replicon RNA into RRPs which is secreted into the culture medium. When RRPs infect BHK-21 cells, the replicon RNA expresses GFP and non-structural proteins which amplify more RNA. However, no WNV structural proteins or additional RRPs produced in RRP-infected cells, thereby preventing further spread. When BWNV-CME cells are infected with RRPs, the structural proteins expressed by the cells package the replicon RNA into progeny RRPs and the infection spreads in rounds similar to the wild type virus.