dNTP pool modulation dynamics by SAMHD1 protein in monocyte-derived macrophages

Abstract

Background: SAMHD1 degrades deoxyribonucleotides (dNTPs), suppressing viral DNA synthesis in macrophages. Recently, viral protein X (Vpx) of HIV-2/SIVsm was shown to target SAMHD1 for proteosomal degradation and led to elevation of dNTP levels, which in turn accelerated proviral DNA synthesis of lentiviruses in macrophages. Results: We investigated both time-dependent and quantitative interplays between SAMHD1 level and dNTP concentrations during multiple exposures of Vpx in macrophages. The following were observed. First, SAMHD1 level was rapidly reduced by Vpx + VLP to undetectable levels by Western blot analysis. Recovery of SAMHD1 was very slow with less than 3% of the normal macrophage level detected at day 6 post Vpx treatment and only~30% recovered at day 14. Second, dGTP, dCTP and dTTP levels peaked at day 1 post Vpx treatment, whereas dATP peaked at day 2. However, all dNTPs rapidly decreased starting at day 3, while SAMHD1 level was below the level of detection. Third, when Vpx pretreated macrophages were re-exposed to a second Vpx treatment at day 7, we observed dNTP elevation that had faster kinetics than the first Vpx + VLP treatment. Moreover, we performed a short kinetic analysis of the second Vpx treatment to find that dATP and dGTP levels peaked at 8 hours post secondary VLP treatment. dGTP peak was consistently higher than the primary, whereas peak dATP concentration was basically equivalent to the first Vpx + VLP treatment. Lastly, HIV-1 replication kinetics were faster in macrophages treated after the secondary Vpx treatments when compared to the initial single Vpx treatment. Conclusion: This study reveals that a very low level of SAMHD1 sufficiently modulates the normally low dNTP levels in macrophages and proposes potential diverse mechanisms of Vpx-mediated dNTP regulation in macrophages.

Metabolic profiling during HIV-1 and HIV-2 infection of primary human monocyte-derived macrophages

“…6B). However, these dNTP increases were much lower in magnitude and shorter in duration as compared to our data using VLPs (Hollenbaugh et al, 2014; Kim et al, 2012). Collectively, HIV-2 infected MDMs do not maintain high levels of dNTPs, since they are only needed during the reverse transcription and gap repair during viral DNA integration (Amie et al, 2013; Van Cor-Hosmer et al, 2013), which are very early events in the viral life cycle.…”

“…5E) remained relatively comparable without increasing from day 0. As expected, these data showed that Vpx in GL-AN virus containing Vpx promoted the increase in dNTPs, but this increase was much more transient in duration as compared to VLP treated MDMs (Hollenbaugh et al, 2014). Collectively, the importance of elevated dNTP concentration is most marked early after infection, when the majority of reverse transcription is thought to take place.…”

“…Our previous studies showed no augmentation of SAMHD1 protein levels in MDMs transduced by single-round D3HIV-1 vector or treatment with virus-like particles not containing Vpx (Hollenbaugh et al, 2014; Kim et al, 2012). Immunoblot analysis (Fig.…”

“…Previously reports have shown that VLPs containing Vpx or HIV-1 engineered with Vpx promote SAMHD1 degradation and led to a rapid increase in dNTPs by 24 h (Berger et al, 2011b; Hollenbaugh et al, 2014; Kim et al, 2012; Sunseri et al, 2011). HIV-1 strains do not contain Vpx; therefore, HIV-1 does not promote rapid degradation of SAMHD1, which would led to the rapid increase dNTP concentration during the first 12 h of infection (Kim et al, 2012).…”

SAMHD1: Recurring roles in cell cycle, viral restriction, cancer, and innate immunity

“…SAMHD1 is widely established as a central regulator of dNTP pool dynamics within cells and as such its catalytic activity can directly influence the replicative capacity of the cell (97, 133–136). Silencing or degradation of SAMHD1 results in elevated dNTP pools and altered growth kinetics as evidenced by studies that demonstrate an increased retention of cells in the G 1 phase with a corresponding loss in S-phase cells (97, 133, 137).…”