FIG 2.

Adaptation of the tripartite GFP assay for analysis of EFC interactions. (A) Construction of VACV-GFP1-9 and VACV-GFP1-10. Infusion PCR was used to insert DNA encoding GFP1-9 or GFP1-10 into the transfer plasmid pRB21 so as to be regulated by the VACV early-late promoter. The resulting GFP1-9 and GFP1-10 plasmids were transfected into BS-C-1 cells that had been infected with vRB12 to allow homologous recombination and large plaque formation. Virus from large plaques was clonally purified by repeat plaque formation to obtain VACV-GFP1-9 and VACV-GFP1-10. (B) Construction of plasmids expressing tagged EFC proteins. GFP10 or GFP11 sequences were fused to DNA encoding individual EFC proteins with an HA or V5 tag at the ectodomain that are regulated by the late p11 VACV promoter. (C) Expression of EFC proteins with V5 tags in VACV-infected cells was demonstrated by Western blotting. Similar results were obtained with HA-tagged EFC proteins (not shown).