Figure 3. Effect of SAMHD1 on lentiviral infection in hydroxyurea-treated cells.

HeLa cells were transduced with lentiviral particles encoding either WT SAMHD1, the indicated SAMHD1 mutants, or an empty vector and selected with puromycin for 48 h. Cells were treated +/− 1mM hydroxyurea (HU) for 4 h prior to infection or dNTP isolation (in continued presence or absence of HU). A) Total cell extracts were separated by SDS-PAGE and subjected to immunoblotting for SAMHD1 and actin as indicated. B) Cellular dNTPs were isolated at time of infection and the amount of dATP present per million cells was determined using a polymerase-based assay. Data are presented as mean and standard deviation of quantitation from at least 3 independently generated cell lines. C, D & E) Cells were infected with increasing volumes of VSV-G pseudotyped SIV-GFP, with or without Vpx, and the percent infection (% GFP-positive cells) was determined by flow cytometry 24 h later. Results in panels A, C, and D show representative results from one of at least 3 independent experiments. Panel E represents the mean and standard deviation from 3 independent experiments performed as in panel D. The maximum amount of infection determined in the empty vector sample was defined as 100% for each experiment. All other data points were normalized accordingly.