Tight interplay among SAMHD1 protein level, cellular dNTP levels, and HIV-1 proviral DNA synthesis kinetics in human primary monocyte-derived macrophages

Abstract

Recently, SAMHD1 has come under intense focus as a host anti-HIV factor. SAMHD1 is a dNTP triphosphohydrolase, which leads to the regulation of DNA metabolism in host cells. HIV-2/SIV (simian immunodeficiency virus) viral protein x (Vpx) has been shown to promote the degradation of SAMHD1. In this study, we examine the kinetics of SAMHD1 degradation, the increase in the dNTP pool level, and the efficiency of proviral DNA synthesis in Vpx+ virus-like particle (VLP)-treated monocyte-derived macrophages (MDMs). Our results indicate a very close temporal link with a reduction in SAMHD1 detected within the first few hours of Vpx+ VLP treatment. This loss of SAMHD1 is followed by a significant increase in cellular dNTP levels by 8 h after Vpx+ VLP addition, ultimately leading to the enhancement of the HIV proviral DNA synthesis rate and HIV infection in MDMs. Finally, the pretreatment of MDMs with the Vpx+ VLPs, which is a widely used protocol, displayed identical proviral DNA synthesis as compared with MDMs co-treated with Vpx+ VLP and HIV vector. These findings further indicate that Vpx degradation of SAMHD1 is sufficiently rapid to enable appropriate progression of reverse transcription in MDMs, even when present at the time of infection. Overall, this study demonstrates a tight interplay between SAMHD1 level, dNTP levels, and HIV proviral DNA synthesis kinetics in MDMs.

FIGURE 1.

Western blot analysis of MDM lysates. A, a representative Western blot is shown for SAMHD1 and actin expression levels. Two million MDMs were treated with VLPs with and without Vpx (145 ng p27/million cells), and then harvested at the indicated time points. B, data are graphed as the mean and S.E. for three independent donors. Data were normalized to actin, used as a loading control. Student's t test analysis was performed to determine statistical significance (p < 0.05) for the different time points.

FIGURE 2.

HIV RT-based primer extension assay. A, a representative HIV-1 RT-based primer extension gel is shown. Extension products are indicated by Primer +1 lane. Extracts from Vpx− VLPs are shown at the top, whereas Vpx+ VLPs extracts are below. B, data collected are graphed as mean and S.E. for three independent donors. Student's t test analysis was performed to determine statistical significance between the different time points.

FIGURE 3.

qPCR analysis for 2LTR circle copy numbers. A, MDMs were pretreated for 2 h with VLPs before the addition of D3-HIV GFP virus. At the indicated time points, cells were collected and processed for total DNA. qPCR for 2LTR circle copy numbers were performed in duplicate for three independent donors. B, MDMs were co-treated with VLPs and D3-HIV GFP virus. Data are plotted as mean and S.E., and paired Student's t test analysis was performed to determine statistical significance.