Figure 8

Over-expression of SAMHD1 in HEK 293T cells or HeLa cells does not inhibit HIV-1 infection, but only modestly decreases the intracellular dNTP pool. (A) HEK 293T and HeLa cells were transiently transfected with SAMHD1-expressing construct or an empty vector. SAMHD1 expression was detected by immunoblotting with SAMHD1-specific antibodies at 24 hr post-transfection. Equal amounts of total protein (20 μg) of the lysates of DCs from two donors and HEK 293T and HeLa cells were loaded. (B-D) HEK 293T cells and (E-G) HeLa cells were transiently transfected with HA-tagged SAMHD1-expressing construct or an empty vector. SAMHD1 expression in transfected HEK 293T cells (B) or HeLa cells (E) was detected by immunoblotting with anti-HA at 24 hr post-transfection. β-actin was used as a loading control. Transfected HEK 293T cells (C) or HeLa cells (F) were infected with HIV-Luc/VSV-G at an MOI of 0.5, and the infection was detected by measuring luciferase activity in the cell lysates at 48 hr post-infection and normalized to protein content (10 μg/sample). cps, counts per second. The intracellular dNTP pool of transfected HEK 293T cells (D) or HeLa cells (G) was measured at 24 hr post-transfection using the single-nucleotide incorporation assay. The asterisks indicate a significant difference (p <0.05) compared with the vector controls. The data shown represents one of two or three independent experiments. Error bars represent standard deviation of the mean of duplicate or triplicate samples.