Fig. 2

Production of MERS VLPs in cultured cells. a Expression of MERS-CoV structural proteins. Plasmids encoding MERS-CoV structural proteins M, E, S, and N as indicated were cotransfected into 293 T cells and the cell lysates were prepared for Western blot analysis two days posttransfection. Protein lysate prepared from cells transfected with control plasmid was applied as a negative control (lane Ctrl). b Electron microscopic analysis of MERS VLPs. The cells were fixed and embedded three days following cotransfection of the plasmids encoding the MERS-CoV structural proteins M, E, and S (VLPdN) or M, E, S, and N (VLP). Images of the sections were examined for the presence of MERS VLPs in a Hitachi H-7100 electron microscope equipped with a Gatan 832 digital camera. c Separation of MERS VLPs. MERS VLPs collected from culture medium were subjected to a discontinuous sucrose gradient centrifugation as described in Methods. The fractions were collected for Western blot analysis. Lane CL represents the protein lysates of transfected cells and lane VLP represents the total VLPs prior to the gradient centrifugation