dNTP pool modulation dynamics by SAMHD1 protein in monocyte-derived macrophages
© Hollenbaugh et al.; licensee BioMed Central Ltd. 2014
Received: 23 April 2014
Accepted: 18 July 2014
Published: 27 August 2014
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.
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.
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.
Sterile alpha motif (SAM) and histidine/aspartic acid (HD) domain protein 1 (SAMHD1) has been linked to Aicardi-Goutières Syndrome, which is a rare autoimmune disease . In addition to its role in autoimmunity -, SAMHD1 has been studied in the context of antiviral response ,- and genomic stability ,. Several groups have now shown that SAMHD1 is found in all cell types and localizes to the nucleus ,,-.
Recent evidence indicates that SAMHD1 has at least two different cellular functions. First, SAMHD1 was shown to have deoxyribonucleoside triphosphate (dNTP) phosphohydrolase activity ,, 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 -. Recently, both biochemical and structural evidence indicated that SAMHD1 forms a tetramer as the active dNTP phosphohydrolase complex -. When dNTPs bind in the active site, the tetramer is formed, and the tetramer was suggested to be long-lived in the cell . SAMHD1 tetramer could maintain the cellular dNTP concentrations at a very low level outside the S phase of the cell cycle. Second, SAMHD1 was shown to have nuclease activity. The nuclease activity was localized to the HD domain of SAMHD1 . Both single-stranded DNA and RNA nuclease activities have been reported for SAMHD1 ,.
Regulation of SAMHD1 occurs by three mechanisms. First, promoter methylation was shown to inhibit transcription , leading to a reduction in SMAHD1 levels. Second, SAMHD1 is regulated during S phase ,, being targeted for degradation. Third, SAMHD1 phosphorylation at T592  was shown to regulate its antiviral activity but not dNTP phosphohydrolase activity . Importantly, White et al. have shown non-dividing cells do not phosphorylate SAMHD1 at T592, whereas cycling cells do .
HIV-2 and some SIV strains encode for the accessory viral protein X (Vpx). It has the ability to target human SAMHD1 to the proteasome for degradation by DCAF1-E3-ubiquitin ligase -. Vpx interacts with the C-terminus of SAMHD1 in order to facilitate this degradation ,,. Recent reports have examined the acute kinetics of Vpx-mediated SAMHD1 degradation in myeloid cells and the enhancement of HIV-1 infection after Vpx treatment ,. Further, we have reported in detail the acute effects of Vpx-mediated SAMHD1 degradation in monocyte-derived macrophages (MDMs) , which led to increased dNTP levels followed by enhancement of proviral DNA synthesis and transduction of MDMs.
In this report we performed an extensive kinetic and quantitative analysis examining the prolonged changes in dNTP concentrations and SAMHD1 levels over 14 days in primary human MDMs. In addition, we treated macrophages either with a single or dual VLP treatments, i.e. after the single Vpx + VLP treatment that kept SAMHD1 levels reduced by Western blots, and then measured dNTP levels and cellular nucleotide metabolites. While SAMHD1 remained very low, a second Vpx + VLP treatment promoted a rapid and robust increase in dNTPs. Collectively these data suggest that a very low level of SAMHD1 can dramatically modulate the dNTP concentrations in primary human MDMs.
Monitoring the long-term kinetics of SAMHDlevels and dNTP concentrations in human primary monocyte-derived macrophages
Next, we employing our highly sensitive HIV-1 RT-based dNTP assay  to examine the effects on dNTP pools after Vpx-mediated SAMHD1 degradation in the same MDM donors used in Figure 1B. As shown in Figures 1D-G, increases in all four dNTPs were clearly detected after Vpx + VLP treatment in MDMs. dATP and dGTP concentrations (Figures 1D and 1E) were significantly increased, while dCTP and dTTP concentrations (Figures 1F and 1G) were only modestly increased. dGTP concentration peaked at day 1, consistent with our published results . Importantly, dATP peaked at day 2 before the contraction began, indicating dNTP modulation was different for each of the nucleotides. The dNTP contraction occurred even though SAMHD1 protein remained undetectable at days 3–5 by Western blot analysis. Basically, the rapid dNTP retraction following its acute elevation by Vpx occurred much ahead of the SAMHD1 recovery. These data suggest that the decrease in dNTP levels may be independent of the total SAMHD1 protein level due to various possibilities, which are discussed below.
Effect of dual Vpx + VLP treatment on dNTP levels in MDMs
Comparison of the effects of the single and dual Vpx + VLP treatments on HIV-transduction efficiency in MDMs
Measurement of dNTP intermediate metabolites by quantitative LC-MS/MS
Effect of gemcitabine, an RNR inhibitor, on the Vpx-mediated dNTP elevation in MDMs
This investigation began by examining how quickly SAMHD1 protein levels return after a single Vpx + VLP treatment in seven day maturated, primary human MDMs. We found that the level of SAMHD1 remained very low (less than 3% of the normal level in macrophages) between days 1-6 before it became consistently detectable by Western blot analysis at day 7. Vpx has been shown to have a long cellular half-life  and should be degraded roughly by day 5 after VLP treatment. However, SAMHD1 never recovered to its normal high level even at day 14, suggesting de novo protein synthesis of SAMHD1 may also be very slow or negatively regulated after Vpx treatment. Examining dNTP levels over this long time course showed a different observation. The levels of dNTPs were reduced well before the detection of SAMHD1 level by Western blot (Figure 1A), yet dNTPs declined starting at day 2 for dGTP, dCTP and dTTP and day 3 for dATP post Vpx + VLP treatment. Thus we speculate that additional factors or very low levels of SAMHD1 may be regulating dNTP pool sizes. We expected that the reduced level of SAMHD1 at day 7 post Vpx + VLP treatment would provide a window of opportunity to investigate cellular dNTP metabolism during a second Vpx + VLP exposure. Indeed, the dual Vpx + VLP treatment of MDMs at day 7 was able to display the same robust dNTP elevation at the time point when SAMHD1 remained less than 3% of the normal level in MDMs (Figure 2). We speculate that since the kinetics of the dual treatment are faster and the HLPC-MS data indicated an increase in dNDP metabolites, Vpx may harbor a SAMHD1 independent function that actively facilitates cellular dNTP biosynthesis metabolism and elevates cellular dNTP levels in the presence of only 3% of the normal SAMHD1 level. This potential mechanism is not related to the up regulations of RNR subunits (Additional file 2). Moreover, we have no direct evidence that Vpx interacts with any of the RNR subunits. Thus, it remains unclear as to just how Vpx VLP treatment and dNDP increase might be occurring.
Interestingly, the deoxypurine triphosphate (dATP and dGTP) concentrations remained high for several days after the single VLP treatment, while deoxypyrimidines – dCTP and dTTP concentrations showed only a transient increase before returning to base line levels for the majority of the MDM donors tested in this study (Figures 1E-F). Our data are consistent with the recently generated SAMHD1 deficient mouse , which shows that dATP and dGTP concentrations were significantly increased in the SAMHD1 deficient mice as compared to wild type mice. Importantly, the dual Vpx + VLP treatment of MDMs was informative by showing that the peak of the dATP and dGTP occurred around 8 h post Vpx + VLP addition, which is much faster than our results published for dGTP on the acute kinetics by the single Vpx + VLP treatment . Interestingly, we observed a decline in the levels of all dNTPs at day 3 post Vpx + VLP treatment, suggesting that turnover of the dNTP pool occurs by other underlying mechanisms other than SAMHD1. These other mechanisms may include hydrolysis by deoxynucleoside diphosphatases ,, shut-off of RNR activity, or conversion to energy currency for other cellular enzyme reactions. Since MDMs are non-dividing cells, we can rule out that the decrease in dNTPs after a dual Vpx + VLP treatment is due to the dNTP utilization during DNA replication. However, we cannot rule out that DNA repair activity is occurring and is consuming the dNTPs. We postulate that the elimination of SAMHD1 may lead to establishing a new modulation set point of dNTP pools within the cell, with dATP and dGTP concentrations remaining much higher than dCTP and dTTP levels .
We employed a series of biochemical and virological investigations with extensive and multiple exposures of Vpx + VLP treatments to human primary MDMs. These studies revealed that there was a significant quantitative discord between levels of total SAMHD1 protein and cellular dNTPs when MDMs were treated with Vpx + VLP. One potential explanation is that Vpx may promote targeting of SAMHD1 for degradation but also facilitate dNTP biosynthesis in macrophages since we detect an increase in dNDP metabolites, which are the precursors for dNTPs. This in turn would achieve a rapid and robust dNTP elevation, which is necessary for accelerating lentiviral reverse transcription for HIV-2 and SIV and also DNA gap-filling repair as part of lentiviral integration , in cells having extremely low cellular dNTP abundance.
These experiments used primary human monocytes obtained from human buffy coats (New York Blood Services, Long Island, NY). These are pre-existing materials that are publicly available, and there is no subject-identifying information associated with the material obtained from this supplier. As such, the use of these samples does not represent human subjects research because: 1) materials were not collected specifically for this study, and 2) we are not able to identify the subjects.
Primary human monocytes were isolated from the peripheral blood buffy coats by positive selection using MACS CD14+ beads as previously described . Monocytes were maturated into monocyte-derived macrophages (MDMs) in the presence of 5 ng/ml hGM-CSF (Miltenyl Biotec) treated at days 0 and 2 of maturation. MDMs were used at day 7 of maturation for experiments.
Primer extension assay
Protocol was followed as previously described . MDMs were lysed with 60% cold methanol. Cellular debris was cleared by 14 K rpm centrifugation. Supernatant was dried using a SpeedVac (Thermo Scientific). Pellets were resuspended in 20 μl water. Two microliters of sample were used in the primer extension assay. 5' 32P-end-labeled primer (“P”; 5’-GTCCCTCTTCGGGCGCCA-3’) was individually annealed to one of four different templates (3`-CAGGGAGAAGCCCGCGGTN-5’). The template:primer complex was extended by HIV-1 reverse transcriptase, generating one additional nucleotide extension product (“P + 1”) for one of four dNTPs contained in the dNTP samples extracted form the cells. In this assay, the molar amount of the P + 1 product is equal to that of each dNTP contained in the extracted samples, which allows us to calculate and compare the dNTP concentrations for the different treatments .
T225 flasks containing 293FT cells were transfected with 40 μg of pVpx- VLP or pVpx + VLP (kindly provided by Drs. Florence Margottin-Goguet and Nathaniel Landau) and 20 μg of pVSVg at a ratio of 1 μg of DNA to 3 μl of polyethylenimine (1 mg/ml). The following day, medium was discarded and replaced with fresh DMEM medium (5% FBS and antibiotics). On days 2-3 after transfection, the medium was collected and replaced with fresh medium. On the day of collection, medium was centrifuged at 1200 rpm for 5 min to remove cells. Supernatant was subsequently filtered through a 0.45-μm membrane (Corning Inc.) and overlaid on top of 5 ml of a 25% sucrose cushion (25% (w/v) sucrose, 10 mM Tris–HCl [pH 7.5], 0.1 M NaCl and 1 mM EDTA). VLPs were concentrated at 28,000 rpm for 90 min by ultracentrifugation. Supernatant was aspirated, and pellets were resuspended in 600 μl of serum-free DMEM. Supernatant was centrifuged for 1 min at 14 K rpm to remove debris. Aliquots (50 μl) were stored at −80°C. The p27 antigen level was determined using an ELISA kit (Advanced BioScience Laboratories, Inc., Rockville MD). A minimum of 145 ng of p27/million cells was used in experiments.
pD3HIV-GFP vector encodes the HIV-1 NL4-3 genome with the eGFP gene in place of the HIV-1 nef gene and has a deleted envelope gene . To generate virus, 293FT cells in T225 flasks were transfected with 60 μg pD3HIV-GFP and 20 μg pVSV-g using 140 μl polyethyenimine (1 mg/ml) in 37 ml DMEM medium/flask. At day 1 of HIV-1 production, medium was discarded and replaced with fresh complete DMEM medium (5% FBS plus antibiotics). At day 2, the medium was harvested and replaced. The medium was centrifuged at 2500 rpm for 7 min to remove cellular debris, and then stored at 4°C in T75 flask. Day 3 medium was harvested and processed as described for day 2. D3HIV-GFP was concentrated using ultracentrifugation (22 K rpm for 2 h in a SW32 Ti rotor). Pellets were resuspended in 0.5 ml serum free DMEM medium. Afterwards, debris was removed by centrifugation (14 K for 2 min). Sample aliquots (50 μl) were frozen at -80°C until used. MDMs were transduced with D3HIV-GFP and then the samples were analyzed using Accuri C6 flow cytometer monitoring GFP expression at the indicated times. Data files were analyzed using FlowJo software (TreeStar).
MDM extracts were generated by scraping wells with 70% methanol and freezing them overnight at -80°C. Extracts were centrifuged at 13,000 × g for 3 min and the supernatants were subsequently dried. The resulting samples were reconstituted in HPLC mobile phase for LC-MS/MS analysis as described previously . In short, samples were reconstituted in 200 μl of 2 mM NH4H2PO4 with 3 mM hexylamine then analyzed for deoxyribonucleosides. Samples were separated using Hypersil Gold 100 1 mm column using Mobile phase - A: acetonitrile and B: 2 mM NH4H2PO4 with 3 mM hexylamine. A increased from 5% to 50% in 10 min, keep 50% for 3 min. The m/z parent to product MS/MS transitions: 523 to 146, 539 to 162, 496 to 119, and 495 to 81 were applied for the standard stable labeled isotopes and 508 to 136, 524 to 152, 484 to 112, and 485 to 81 for the corresponding sample nucleotides, respectively.
Samples were reconstituted in 200 μl of 2 mM NH4H2PO4 with 3 mM hexylamine, and then split into two fractions. One fraction was analysis for deoxyribonucleosides. Samples were separated using Hypersil Gold 100 × 1 mm column using Mobile phase - A: acetonitrile and B: 2 mM NH4H2PO4 with 3 mM hexylamine. A increased from 5% to 50% in 10 min, keep 50% for 3 min. Instrument parameters were optimized for each metabolite (Additional file 5).
Western blot analysis
Samples were processed in RIPA buffer containing 1 μM DTT, 10 μM PMSF, 10 μl/ml phosphatase inhibitor (Sigma) and 10 μl/ml protease inhibitor (Sigma). The cells were sonicated with 3×, 5 second pulses, to ensure complete lysis. Cellular debris was removed by 15 K rpm centrifugation for 10 min. Supernatants were stored at -80°C before use. Cell lysates were resolved on a 8% SDS-PAGE gel. Proteins were transferred to nitrocellulose membrane and detected as described in the figure legends using the following antibodies: rabbit anti-SAMHD1 mAb (Abcam), anti-GAPDH mouse mAb (Santa Cruz). Anti-mouse and anti-rabbit secondary HRP antibodies were purchased from GE HealthScience. HRP was detected using chemiluminescent reagents (Pierce) following the manufacturers instructions. Images were captured using BioRad ChemiDoc Imager. Anti-pSAMHD1 T592 antibody was obtained from Dr. Diaz-Griffero.
Graphing and statistical analysis
Prism software was used for plotting the data. All the data sets were compared for significant difference using two-way ANOVA analysis and Bonferroni post-test analysis for significance.
We would like to thank Waaqo Daddacha and Laura Nguyen for technical assistance with the HIV-1 RT-based dNTP assay. We would also like to thank Michael Hirschman and Michele Daly for technical assistance. This study was supported by NIH AI049781 (B.K.), GM104198 (B.K.), 5P30-AI-50409 Emory Centers for AIDS Research (CFAR), AI087390 (F. D-G), and the Department of Veterans Affairs.
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