Abstract
BackgroundSAMHD1 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.ResultsWe 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.ConclusionThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0063-2) contains supplementary material, which is available to authorized users.
Highlights
SAMHD1 degrades deoxyribonucleotides, suppressing viral DNA synthesis in macrophages
Monitoring the long-term kinetics of SAMHD1 levels and deoxyribonucleoside triphosphate (dNTP) concentrations in human primary monocyte-derived macrophages We previously reported the acute effects of viral protein X (Vpx) containing virus-like particles (VLP) treatment on human primary MDMs out to 48 h [42] and observed that the dGTP levels had peaked at day 1 and already started to decline at day 2 post Vpx treatment, while the SAMHD1 protein remained undetectable by western blot analysis
Effect of dual Vpx + VLP treatment on dNTP levels in MDMs The day 7 time point after single Vpx + VLP treatment of MDMs (Figure 1B) provided us with the unique opportunity to test whether dual (2×) Vpx + VLP treatment influences the dNTP concentrations when SAMHD1 was less than 5% of the normal SAMHD1 level
Summary
SAMHD1 degrades deoxyribonucleotides (dNTPs), suppressing viral DNA synthesis in macrophages. SAMHD1 was shown to have deoxyribonucleoside triphosphate (dNTP) phosphohydrolase activity [20,21], suggesting it is a host antiviral restriction factor to limit replication of retroviral and DNA containing viruses by depleting cellular dNTPs in viral non-dividing target cell types [22,23,24,25]. Both biochemical and structural evidence indicated that SAMHD1 forms a tetramer as the active dNTP phosphohydrolase complex [26,27,28,29]. Both single-stranded DNA and RNA nuclease activities have been reported for SAMHD1 [29,32]
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