Figure 3.

The R5 pre-AIDS HIV-1 clone 32D2 Nef protein enhances infectivity more than the R5 AIDS allele. The 5BD.1 HIV-1 vector packaging cell line was co-transfected with pTR167 ΔNef (5 μg), pCMV-Tat (2 μg) and *E11, 32D2, NL4-3, or 8G9 pCMV Nef plasmid (2.5 μg). Three days post-transfection, 100 μl of the cell supernatants were used to infect 2 × 10⁵ HeLa CD4 cells in the presence of 8 μg/ml DEAE-dextran. Viral vectors and cells were incubated together at 37°C for 24 hours. At that time, infectious media was removed and replaced with IMDM plus 10% BCS. At 48 hours post-infection, IMDM + 10% BCS and hygromycin (200 μg/ml) was added. After two weeks of selection, the resultant colonies were stained with crystal violet and manually counted. The average of nine infections from two different viral vector stocks for each Nef is shown. Error bars represent standard errors of the mean (SEM). Asterisked 32D2 samples were significantly different from each of the three other Nef positive samples by the Student's unpaired t-test.