Figure 3

Mutation of the YW motif in F13L blocks association of p37-GFP with TIP47 and prevents complementation of an F13L-deleted virus. (A) Diagram of the VV F13L gene with functional domains indicated. (B) Complementation experiment demonstrating a panel of mutant F13L alleles containing the indicated A or F substitutions. The F13L allelescontained a GFP tag at the C-terminus to facilitate immunoprecipitation of p37. PCR products containing the indicated F13L allele were transfected into BSC-40 cells infected with an F13L-deletion virus. At 3 days post infection/transfection, plaques were visualized by staining with crystal violet, (C) Membrane fractions, prepared as described in Materials and Methods, from infected and transfected cells were immunoprecipitated in hypotonic buffer with anti-GFP antibody and probed with TIP47 antibody (lower panel). The upper panel shows the input controls.