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. 2007 Jan 3;104(2):624–629. doi: 10.1073/pnas.0606730104

Fig. 3.

Fig. 3.

Nucleocapsid association of and transcription activation by substitution mutants of VP30. (A) Colocalization studies of NP with VP30 substitution mutants. Plasmids encoding NP and VP30 substitution mutants were transfected into HUH7 cells. At 16 h after transfection, cells were fixed, and immunofluorescence analysis was performed by using a mouse monoclonal anti-VP30 IgM (dilution 1:10) and a mouse monoclonal IgG anti-NP antibody (dilution 1:20). Bound antibodies were detected with FITC-labeled goat anti-mouse IgG serum (Dianova; dilution 1:200) and rhodamine-labeled goat anti-mouse IgM antibody (Dianova; dilution 1:100). (B) Coimmunoprecipitation analysis of VP30 and VP30 mutants with NP. HUH7 cells were cotransfected with plasmids encoding wild-type VP30 or VP30 mutants and NP. Cells were metabolically labeled and immunoprecipitated as described in Experimental Procedures. Immune complexes were separated by SDS/PAGE and exposed to a Bio-Imager (Fuji, Düsseldorf, Germany) plate. The positions of VP30 and VP30 mutants and cosedimented NP are shown by arrows. (C) Incorporation of VP30 substitution mutants into VLPs. HEK 293 cells were transfected with plasmids encoding all structural proteins of EBOV, a minigenome with the Renilla luciferase reporter gene, the T7 DNA-dependent RNA polymerase, and the firefly luciferase. If indicated, plasmid-encoding VP30 was replaced by VP30 mutants. Released VLPs were purified through a 20% sucrose cushion and treated with proteinase K for 1 h. Digestion was stopped with PMSF and samples were separated by 12% SDS/PAGE and blotted onto a nitrocellulose membrane. Immunostaining was performed with a mouse anti-flag antibody (dilution 1:15,000) and a mouse anti-NP antibody (dilution 1:200). Bound antibodies were detected with an IRDye700DX goat anti-mouse antibody (Rockland, Gilbertsville, PA; dilution 1:5,000) and analyzed with LiCor Odyssey Infrared Imaging System (Li-Cor Biosciences, Bad Homburg, Germany). (Upper) Proteinase K digested VLPs. (Lower) Quantification of wild-type VP30 or VP30 mutants incorporated into VLPs. Incorporation of VP30 and VP30 mutants was normalized against the incorporated amount of NP. Incorporation of wild-type VP30 was set to 100%. (D) Analysis of EBOV-specific transcription activation by VP30 and VP30 substitution mutants. HEK 293 cells were transfected with plasmids expressing the nucleocapsid proteins of EBOV, an EBOV-specific minigenome containing the Renilla luciferase reporter gene, and the T7 DNA-dependent RNA polymerase. As control for cellular transcription, the vector pGL2 was transfected, which contained the gene for the firefly luciferase under a simian virus 40 promoter. At 3 days after transfection, cells were lysed, and activities of Renilla luciferase and firefly luciferase were measured. Renilla luciferase activity was normalized by firefly luciferase activity, and the activity gained with wild-type VP30 was set to 100%.