FIG 3.

Effect of L72A substitution on Z's functions. (A) Effect of L72 substitutions on Z-mediated budding. 293T cells were transfected with WT or the indicated mutant Z protein-expressing plasmids. At 48 h posttransfection, VLPs present in tissue culture supernatants were collected by ultracentrifugation and total cell lysates prepared. Levels of Z protein in VLPs and cell lysates were determined by Western blotting using an antibody to Strep. (B) Effect of L72 substitution on Z's ability to inhibit MG activity. 293T cells were transfected with pT7-MG/eGFP and pC-T7pol, pC-NP, and pC-L, together with or without [Z(−)], a plasmid expressing the WT or the indicated mutant Z proteins. For a negative control, pC-L was omitted from the transfection mix [L(−)]. At 72 h posttransfection, eGFP expression was examined by epifluorescence (i); whole-cell lysates were prepared and Z protein expression levels in clarified cell lysates were examined by Western blotting using an antibody to Strep (ii).