Impact of V66A, G67A and F68A mutations on Nef-mediated enhancement of virion infectivity. (A), HeLa-MAGI cells were infected with HIV-1SF2 and HIV-1SF2 with mutant nefs from Figure 4. ∆Xho represents HIV-1SF2 with its nef rendered non-functional by filling in the unique XhoI site with Klenow to make a four base insertion. β-galactosidase positive cells were counted 36 hours post-infection. Viral infectivity (blue cells per ng p24gag normalized to HIV-1SF2) are presented as the mean ± S.E.M. with n = 6. SF2Nef was set at 100%. An asterisk indicates a significantly lower value compared to SF2Nef by t test (p < 0.05). Also note that values for all mutated proteins were significantly higher than ∆Xho. (B), Lysates from 293T producer cells for the viruses in (A) were analyzed for Nef and p24gag (p24) expression by Western blot. Equal amounts of total lysate protein were added to each lane.