Fig. 7

SAMHD1 dephosphorylation at T592 upon G₁ entry correlates with a decrease in HIV-1 RT products in activated primary CD4⁺ T cells. a Schematic of infection experiment in activated primary CD4⁺ T cells. After activation for 5 days by PHA-P/IL-2, primary CD4⁺ T cells were infected with VSV-G-pseudotyped HIV-1 reporter virus (-/+ Vpx) for 2 h. After removal of the virus, cells were arrested in mitosis by nocodazole (N) treatment. Subsequently, CD4⁺ T cells were washed twice 18 h post-infection (p.i.) and, as a result, upon removal of nocodazole cells could progress to G₁ phase (condition “inf”), whereas upon continuation of nocodazole treatment, cells remain in mitosis (condition “inf + N”). Total DNA for RT product measurements was harvested 24 h p.i; MOI 7.5). Importantly, the time of removal or continuation of nocodazole was chosen in such a way that sufficient DNA copies at 16 h p.i. could be detected and coincided with specific cell cycle-phases. b, d Increased SAMHD1 phosphorylation at T592 after mitotic arrest correlates with higher HIV-1 RT copy numbers. Primary CD4⁺ T cells were infected with VSV-G-pseudotyped HIV-1 luciferase reporter virus (-/+ Vpx) or heat-inactivated virus and subsequently treated with nocodazole as described in Fig. 7a. 24 h p.i., total DNA was collected and the amount of early (b) and late (d) RT products determined by qPCR. Each sample was measured in technical triplicates. Data shown represent the mean ± SD of four donors analyzed (each depicted by a specific symbol). c, e Increase in early (c) and late (e) HIV-1 RT products with (continued) nocodazole treatment depends on the absence of Vpx, correlating with phosphorylated SAMHD1 (Fig. 7f). Differences in RT products ( = Δ(inf + N – inf)) observed in Fig. 7b and d, respectively, after infection with VSV-G-pseudotyped HIV-1 reporter virus in the absence and presence of Vpx were calculated (−/ + Vpx). Statistical significance was determined using a paired, two-tailed Student’s t-test (ns: p ≥ 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001). Data shown represent the mean difference ± SD of four donors analyzed (each depicted by a specific symbol). f Validation of SAMHD1 pT592-status and degradation of SAMHD1 upon Vpx delivery in activated primary CD4⁺ T cells (related to Fig. 7b-e). For immunoblotting, primary CD4⁺ T cells were harvested after activation for 5 days ( = a), after release from nocodazole arrest ( = -N; 24 h p.i.) and with continued nocodazole treatment ( = + N; 24 h p.i.). Whole-cell lysates were analyzed using antibodies specific to the indicated proteins. Data shown represent four donors analyzed