Table 1.

BiFC analysis of Nef dimerization interface mutants. 293T cells were co-transfected with paired BiFC expression vectors for wild-type Nef as well as each of the dimerization interface mutants shown. Forty-eight hours later, cells were immunostained for Nef protein expression and the relative intensities of the BiFC and immunofluorescence signals were quantified using MetaMorph software. Each Nef BiFC signal was then normalized to the Nef protein level as determined by immunofluorescent staining. The percent decrease in BiFC relative to wild-type Nef was then calculated by dividing the normalized BiFC signal for each mutant by the wild-type value, subtracting the result from 1.0, and multiplying by 100. This experiment was repeated in quadruplicate with comparable results in each case. While all mutants decreased Nef-BiFC, the most dramatic decreases were consistently observed with the 4D and D123N mutants (bold).

Nef Mutant	% Decrease in BiFC Relative to WT
L112A	52.8
F121A	33.0
L112D	47.5
Y115D	59.1
L112D/Y115D (DD)	86.7
I109D/L112D/Y115D/F121D (4D)	99.9
D123A	69.6
D123N	97.1
D123V	52.9
R105E	49.9
R105E/R106E (2RE)	71.1
G2A (myristoylation defective)	0.0
2PA (SH3-binding motif)	0.0