Fig. 1.

Construction of a VACV strain WR lacking three innate immunomodulators. (a) Schematic representation of the order in which the C6L and K7R genes were removed from a virus already lacking N1L (vΔN1). Revertant viruses were constructed at each stage as controls. (b) PCR analysis of proteinase K-treated lysates of BSC-1 cells infected with the indicated viruses for 16 h at 2 p.f.u. per cell using primers specific for N1L, C6L, K7R and A49R. (c) Immunoblot analysis of BSC-1 cells infected for 16 h with the indicated viruses at 2 p.f.u. per cell. Lysates were analysed by SDS-PAGE and immunoblotted using monoclonal antibodies against α-tubulin and D8 or with polyclonal antisera against N1, C6 and K7. Molecular mass markers are indicated on the right (kDa). (d) Monolayers of BSC-1 cells were infected with the indicated viruses for 72 h. Cells were stained with crystal violet and the size of 20 plaques was measured using Axiovision acquisition software and a Zeiss AxioVert.A1 inverted microscope. Results are expressed as the mean plaque radius ± SD. Data are representative of at least 2 independent experiments.