FIG. 1.

Production of Ebola VLPs from plasmids. (A) Schematic diagram of the construct for production of the Ebola virus minigenome, p3E5EGFP. This construct was made from plasmid 3E-5E, which contains the chloramphenicol acetyltransferase (CAT) gene in antisense orientation between the leader and trailer sequences of the Ebola virus genome, flanked by the T7 RNA polymerase promoter (T7) and a ribozyme (Rib) (10). The CAT gene was replaced with the GFP gene using NotI and NdeI restriction enzyme sites; thus, the GFP gene is also cloned in the antisense orientation, as indicated by the inverted letters. (B) Schematic diagram of a system for Ebola VLP generation. 293T cells were transfected with plasmids for the expression of the Ebola virus NP, L, VP35, VP30, GP, VP40, and VP24 proteins and with p3E5EGFP. pC-T7Pol was also cotransfected into cells to provide T7 RNA polymerase. Culture supernatants of 293T cells were then harvested 72 h after transfection and were incubated with 293T cells which had been transfected with plasmids for expressing L, NP, VP35, and VP30 proteins 24 h prior to inoculation with the culture supernatant.