Fig. 3
Functional analysis of the putative MERS-CoV RNA packaging signal. a-b Expression of MERS-CoV structural proteins in transfected cells and VLPs. Western blot analysis was performed with the cell lysates (panel a) and both the cellular and supernatant VLPs (panel b) following cotransfection of the plasmids encoding the viral structural proteins and a heterologous GFP-PS mRNA as indicated. The secondary structure of the PS258(19712–19969)ME RNA fragment is shown with the 94-nt stable substructure highlighted as marked in Fig. 1b. c Functional analysis of RNA packaging signal. MERS VLPs were collected from the medium of the cultured cells following cotransfection of the plasmids encoding the viral structural proteins S, E, M, and N, and the GFP vector control plasmid pEGFP-N1 (indicated by VLP), the putative MERS-CoV packaging signal plasmid pEGFP-PS258(19712–19969)ME (indicated by VLP/PSME), and the SARS-CoV packaging signal plasmid pEGFP-N1-PS580 (indicated by VLP/PSSA). VLPdN/PSME represents MERSVLPs produced by cotransfection of the plasmids encoding MERS-CoV M, E, and S proteins and plasmid pEGFP-PS258(19712–19969)ME. These MERS VLPs harvested from different cell sources were incubated with naïve Huh7 cells for 48 h. The treated Huh7 cells were then fixed for immunofluorescence staining with GFP antibody and Hoechst