Figure 5

Inhibiting reverse transcription does not affect the RNase activity of SAMHD1. (a) An in vitro RNase assay was performed using 20mer ssRNA substrates. Purified GST-tagged SAMHD1^WT protein was pre-incubated with 0, 5, or 10 μM d4T (left) or DDI (right), or with 10 μM nevirapine. The reaction was performed in the presence of RNA substrates for 30 min at 37°C. The reaction was stopped by heating to 95°C for 10 min. b shLuciferase THP-1 and shSAMHD1 THP-1 cells were differentiated with PMA and then pre-incubated with nevirapine for 24 h, if required. The cells were then infected with HIV-1-GFP (60 ng per 6 × 10⁵ cells) for 2 h. At the indicated times, viral RNA levels were analyzed by qRT-PCR using gfp-specific primers. c Wild-type SAMHD1-expressing U937 cells were differentiated with PMA and then infected with HIV-1-GFP (10 ng per 1 × 10⁵ cells) or HIV-1^{D185A/D186A/D443N} (10 ng per 1 × 10⁵ cells). Viral RNA levels were monitored by qRT-PCR using gfp-specific primers. In b, c, data were normalized against an endogenous β-actin control. Data are expressed as the mean ± SD from three independent experiments in triplicate. *p < 0.05 compared with the shLuciferase control or the mock-transfected control at 1 h post-infection (two-tailed Student’s t test). ns not significant.