SAMHD1 restricts HIV-1 infection in dendritic cells (DCs) by dNTP depletion, but its expression in DCs and primary CD4+T-lymphocytes cannot be upregulated by interferons
- Corine St Gelais1,
- Suresh de Silva†1,
- Sarah M Amie†2,
- Christopher M Coleman1,
- Heather Hoy1,
- Joseph A Hollenbaugh2,
- Baek Kim2Email author and
- Li Wu1, 3Email author
© St Gelais et al.; licensee BioMed Central Ltd. 2012
Received: 14 October 2012
Accepted: 29 November 2012
Published: 11 December 2012
SAMHD1 is an HIV-1 restriction factor in non-dividing monocytes, dendritic cells (DCs), macrophages, and resting CD4+ T-cells. Acting as a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, SAMHD1 hydrolyzes dNTPs and restricts HIV-1 infection in macrophages and resting CD4+ T-cells by decreasing the intracellular dNTP pool. However, the intracellular dNTP pool in DCs and its regulation by SAMHD1 remain unclear. SAMHD1 has been reported as a type I interferon (IFN)-inducible protein, but whether type I IFNs upregulate SAMHD1 expression in primary DCs and CD4+ T-lymphocytes is unknown.
Here, we report that SAMHD1 significantly blocked single-cycle and replication-competent HIV-1 infection of DCs by decreasing the intracellular dNTP pool and thereby limiting the accumulation of HIV-1 late reverse transcription products. Type I IFN treatment did not upregulate endogenous SAMHD1 expression in primary DCs or CD4+ T-lymphocytes, but did in HEK 293T and HeLa cell lines. When SAMHD1 was over-expressed in these two cell lines to achieve higher levels than that in DCs, no HIV-1 restriction was observed despite partially reducing the intracellular dNTP pool.
Our results suggest that SAMHD1-mediated reduction of the intracellular dNTP pool in DCs is a common mechanism of HIV-1 restriction in myeloid cells. Endogenous expression of SAMHD1 in primary DCs or CD4+ T-lymphocytes is not upregulated by type I IFNs.
KeywordsHIV-1 restriction SAMHD1 Dendritic cells Interferon Intracellular dNTPs
Myeloid lineage cells such as monocytes, macrophages and dendritic cells (DCs) are important immune cells that elicit innate and adaptive immune responses to a variety of pathogens, including viruses. HIV-1 is known to replicate poorly in myeloid cells; however, these cells play an important role in promoting dissemination of HIV-1 to CD4+ T-lymphocytes, the major target of HIV-1 infection [1, 2]. In contrast to HIV-1, HIV-2 and simian immunodeficiency virus (SIV) from the sooty mangaby lineage are able to efficiently infect myeloid lineage cells by a mechanism initially attributed to the Vpx protein mediating degradation of an unknown host cellular restriction factor . Restriction factors are a group of cellular proteins that can block viral replication in cells and are typically upregulated by type I interferons (IFNs) [4–6]. Well-characterized HIV-1 restriction factors include apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) , tripartite motif 5α (TRIM5α) , and tetherin (also known as BST-2 or CD317) [9, 10].
SAM domain and HD domain-containing protein 1 (SAMHD1) was initially identified in myeloid cells as an HIV-1 restriction factor that was degraded by HIV-2/SIV Vpx [11–13]. Recent studies also revealed that SAMHD1 restricts HIV-1 infection in resting CD4+ T-cells [14, 15]. Vpx-mediated degradation of SAMHD1 was found to relieve HIV-1 restriction in myeloid cells and resting CD4+ T-cells, allowing enhancement of HIV-1 infection [11–18]. SAMHD1 is a dGTP-regulated deoxynucleoside triphosphate (dNTP) triphosphohydrolase that hydrolyzes dNTPs in vitro[19, 20]. SAMHD1-mediated HIV-1 restriction occurs via endogenous SAMHD1 depleting the intracellular dNTP pool, thereby inhibiting HIV-1 reverse transcription and viral infection [14, 16, 18, 21]. However, the intracellular dNTP pool concentration in DCs has not been reported. Moreover, the effect of SAMHD1 on regulating the dNTP pool in DCs remains to be investigated.
Mutations in SAMHD1 are associated with a rare genetic disorder known as Aicardi-Goutieres syndrome (AGS) with symptoms of a congenital viral infection likely due to excessive production of IFN-alpha (IFNα) and increased immune activation [22, 23], suggesting that SAMHD1 may act as a negative regulator of the type I IFN response. SAMHD1-deficient CD14+ monocytes and resting CD4+ T-lymphocytes from AGS patients are highly susceptible to HIV-1 infection in vitro[13–15], suggesting that SAMHD1 may be critical for inhibiting HIV-1 infection in vivo. Moreover, IFNα treatment of monocytes isolated from a healthy donor up-regulated the levels of SAMHD1 protein , indicating that SAMHD1 is type I IFN-inducible in monocytes. However, it remained unknown whether endogenous SAMHD1 expression in DCs and primary CD4+ T-cells can be upregulated by type I IFNs.
In this study, we sought to understand the mechanism of SAMHD1-mediated HIV-1 restriction by characterizing SAMHD1 protein expression and response to IFNs in both DCs and primary CD4+ T-lymphocytes. We show that Vpx-mediated degradation of SAMHD1 in DCs significantly enhances HIV-1 infection and accumulation of late reverse transcription products, and increases the intracellular dNTP pool. We observed that endogenous SAMHD1 in primary DCs or CD4+ T-lymphocytes are not readily upregulated by type I IFNs. Our results provide new information on the cellular mechanism of SAMHD1-mediated HIV-1 restriction in DCs and the function of SAMHD1 as an HIV-1 restriction factor.
Vpx-mediated SAMHD1 degradation in DCs significantly increases HIV-1 infection and accumulation of late reverse-transcription products
Treatment of DCs, monocytes, macrophages, and differentiated monocytic cell lines with Vpx-containing SIV virus-like particles (VLPs) has been shown to enhance HIV-1 infection in these cells [11–13, 16, 24–30]. Vpx-mediated degradation of SAMHD1 accounts for the enhanced HIV-1 infection in myeloid cells [11–13, 16]. We confirmed that Vpx-mediated degradation of SAMHD1 enhanced both single-cycle and replication-competent HIV-1 infection in DCs. DCs were transduced with either Vpx-negative SIV VLPs or VLPs containing Vpx from HIV-2 or SIV. These VLPs lack viral genetic material and can be used to effectively deliver Vpx protein to DCs [3, 31]. Mock-transduced DCs served as an additional negative control to exclude any effect of other SIV proteins on SAMHD1 expression. VLP-transduced DCs were infected with single-cycle or replication-competent HIV-1, and viral infection was measured by detecting luciferase activity and p24 capsid release, respectively.
Furthermore, we observed that Vpx-mediated SAMHD1 degradation increased infection of DCs with R5-tropic, replication-competent HIV-1NLAD8, as evidenced by 4-fold enhancement of p24 production in the supernatants from infected DCs at 72 hr post-infection (p <0.001) (Figure 1E). Immunoblotting of DC lysates from the same experiment indicated that de novo production of HIV-1 Gag p55 was significantly enhanced in SAMHD1 knockdown cells compared to negative controls (Figure 1F), confirming productive infection. Similar results were obtained using VLPs containing endogenous SIVmac251 Vpx (Vpx is expressed from the SIVmac251 proviral DNA rather than trans-complemented) (data not shown). These results demonstrate that Vpx-mediated SAMHD1 degradation efficiently enhances HIV-1 infection of DCs and demonstrates that knockdown of SAMHD1 in DCs enables accumulation of full-length viral cDNA.
Vpx(+) VLP treatment increases the intracellular pool of dNTPs in DCs
The intracellular dNTP concentrations of DCs have not been previously published, and it is not known if SAMHD1 modulates the dNTP pool in primary DCs. To address these questions, we determined the intracellular dNTP levels of DCs and quantified the effect of Vpx treatment on the dNTP pool in DCs using a single nucleotide incorporation assay. DCs from two independent donors were treated with SIV VLPs (+/−) Vpx. Cell samples were harvested at 0, 12, and 24 hr post-VLP treatment and intracellular dNTP levels were determined.
A representative gel image of HIV-1 RT-based single nucleotide primer extension assay is shown for dATP extension (Figure 2B). Extended primer (P+1) can be visualized with increasing intensity in both donor 1 and donor 2 samples in the presence of Vpx at 12 and 24 hr post VLP treatment. These data suggest that primary DCs possess low levels of intracellular dNTPs, in keeping with other non-dividing primary cells, such as macrophages and resting CD4+ T-lymphocytes [14, 21, 37–39]. Our results also suggest that SAMHD1 plays an important role in maintaining the intracellular dNTP pool in DCs.
Type I and type II IFN treatment does not upregulate SAMHD1 protein expression in DCs
Type I and type II IFN treatment causes a transient increase in SAMHD1 mRNA levels at early time points in primary DCs
Kinetics analysis of SAMHD1 protein and mRNA in DCs treated with IFNα
SAMHD1 levels in primary CD4+T-lymphocytes are not upregulated by type I IFN treatment
Type I IFN treatment upregulates SAMHD1 protein expression in HEK 293T cells and HeLa cells
Over-expression of SAMHD1 in HEK 293T cells or HeLa cells does not inhibit HIV-1 infection, but modestly decreases the intracellular dNTP pool in HeLa cells
We next determined the effect of SAMHD1 over-expression on the intracellular dNTP pool and VSV-G-pseudotyped single-cycle HIV-1 infection. SAMHD1 over-expression in HEK 293T and HeLa cells was confirmed by immunoblotting (Figure 8B and 8E). Transfected cells were either infected with VSV-G-pseudotyped HIV-1 and lysates measured for luciferase activity to determine HIV-1 infection or, cellular extracts were processed for dNTP quantification. Even in the presence of high SAMHD1 expression levels, there was no effect on HIV-1 infection in either HEK 293T cells or HeLa cells at 2 days post-infection (Figure 8C and 8F). Interestingly, SAMHD1 over-expression in HEK 293T cells had little effect on the dNTP concentration, at most a 1.3-fold decrease was observed for dTTP levels (Figure 8D). Comparatively, SAMHD1 over-expression in HeLa cells had a more pronounced effect on dNTP concentration and 1.8-, 2.4-, 1.5-, and 2.2-fold decreases were observed for dATP, dCTP, dGTP, and dTTP, respectively (p <0.05) (Figure 8G). These results indicate that SAMHD1 retains its enzymatic activity and is able to modestly hydrolyze dNTPs in HeLa cells; however, the effect is not significant enough to deplete the intracellular dNTP pool to a level that is capable of restricting HIV-1 infection.
In the current study, we examined the role of SAMHD1 in restricting HIV-1 infection of DCs and compared SAMHD1 expression levels following treatment with IFNs. Our results show that Vpx-mediated degradation of SAMHD1 in DCs can relieve a post-entry restriction block against HIV-1 by increasing the intracellular dNTP pool and promoting the accumulation of HIV-1 late reverse transcription products. However, early reverse transcription products were not affected by SAMHD1 degradation, consistent with previous findings in macrophages , suggesting that SAMHD1-mediated HIV-1 restriction mainly affects late reverse transcription. A previous study found that the HIV-1 genome is not completely reverse transcribed in quiescent lymphocytes, unlike in activated lymphocytes . It is likely that HIV-1 early reverse transcription can be initiated in non-cycling cells with low dNTP concentrations, but the late reverse transcription cannot be completed. We also observed that there was no direct correlation between fold increase in HIV-1 late reverse transcription products and fold change in viral infectivity. A potential explanation for the difference is that HIV-1 late reverse transcription product levels may not fully reflect the efficiency of viral gene expression given the complexity of the viral life-cycle in DCs [33–35].
SAMHD1 functions as a dGTP-dependent phosphohydrolase [19, 20], and its degradation with Vpx treatment in DCs increased accumulation of HIV-1 late reverse transcription products, suggesting that SAMHD1 regulates intracellular dNTP levels in DCs. We show that DCs contain low levels of dNTPs (~11–415 nM), within the range of resting T-lymphocytes (300–5,000 nM) , but below that in HIV-1 permissive cell types, such as activated peripheral blood mononuclear cells (PBMCs) (1.5–9.2 μM)  and activated CD4+ T-lymphocytes (3–30 μM) . It appears that DCs have dNTP levels ~1.9- to 2.3-fold higher than macrophages (~50 nM) .
Although HIV-1 infection of DCs is enhanced in the presence of Vpx, the levels of p24 released into the supernatant from infected DCs are lower compared to those from macrophages and fully permissive target cells [32, 33]. This suggests that SAMHD1 has a role in HIV-1 restriction in DCs, but it is likely that additional post-entry restriction steps exist to block HIV-1 infection in DCs. For example, APOBEC3A is highly expressed in myeloid-lineage cells and interacts with Vpx, leading to its degradation, which correlates with increased HIV-1 infection in primary monocytes . Silencing of APOBEC3A relieves restriction of HIV-1 in macrophages, DCs and monocytes , and abolished deaminase activity of APOBEC3A in monocytes . There also remains an unidentified cryptic sensor for HIV-1 infection in DCs dependent on newly synthesized viral capsid .
SAMHD1 restricts HIV1 infection in resting CD4+ T-lymphocytes by limiting reverse transcription through depleting intracellular dNTP concentrations . Previous studies measuring dNTP levels in resting T-lymphocytes suggest that the intracellular dNTP pool is sufficiently low to restrict HIV-1 reverse transcription, which can be attributed to SAMHD1 activity [37, 38]. Recent studies showed that T-cell activation does not significantly affect SAMHD1 expression in primary CD4+ T-cells treated with PHA or with anti-CD3/anti-CD28 for 2–3 days [14, 15]. In agreement with these results, we observed that PHA-treatment of resting CD4+ T-cells for 2 days only slightly decreased SAMHD1 expression in activated CD4+ T-lymphocytes (by 10%- 30% in two donors, Figure 6). Activated CD4+ T-cells have a 3- to 8-fold higher dNTP concentration relative to resting CD4+ T-cells, while SAMHD1 expression remains the same in resting and activated CD4+ T-cells . It is possible that intracellular dNTP levels can be significantly increased when CD4+ T-cells are activated and become dividing cells. How activated CD4+ T-cells upregulate the intracellular dNTP pool without decreasing SAMHD1 expression remains to be investigated.
We found that over-expression of SAMHD1 in dividing cell lines does not restrict HIV-1, similar to a study which found that SAMHD1 expression in dividing cell lines did not have an inhibitory effect on a range of viruses, including HIV-1 . Our dNTP analysis in HeLa cells suggests that SAMHD1 is able to moderately deplete the dNTP pool; but the concentration of dNTPs was within the range of activated T-lymphocytes , suggesting dividing cell lines are capable of maintaining their dNTP pools in the presence of high levels of SAMHD1. It is possible that the catalytic activity of over-expressed SAMHD1 in HEK 293T and HeLa cells may be less stoichiometrically active than the endogenous protein in DCs, and/or that the transformed cell lines lack a potential cellular co-factor(s) for SAMHD1-mediated HIV-1 restriction function .
Analysis of SAMHD1 after IFN treatment indicated that neither type I nor type II IFN treatment affected SAMHD1 protein levels in DCs or primary CD4+ T-lymphocytes. However, analysis of SAMHD1 mRNA levels at 6 hr post-treatment with IFN indicated a 2- to 4-fold increase in mRNA, suggesting that in DCs SAMHD1 is IFN sensitive, albeit transiently. Comprehensive analysis of the effect of IFNα treatment of DCs from 6 to 72 hrs indicated no change in SAMHD1 protein levels and a small transient increase in mRNA levels at 6 and 12 hr post-treatment. Furthermore, our data for IFN treatment of HEK 293T and HeLa cells, as well as previous studies  show that SAMHD1 is type I IFN inducible. Although Berger et al. observed increased SAMHD1 protein levels in primary monocytes upon IFNα treatment, we observed that primary monocytes express lower levels of SAMHD1 relative to DCs (data not shown), which could partially explain the difference in response to IFNα treatment across the two cell types. As we show that DCs have low levels of dNTPs, it is plausible that SAMHD1 expression and/or its activity is tightly regulated in these cells to ensure a minimal dNTP pool is maintained without causing detrimental effects on the cell, for example, DNA repair within cells requires carefully modulated dNTP levels [47, 48]. It is also possible that post-transcriptional regulation of SAMHD1 mRNA may affect SAMHD1 protein expression in the cell. A recent study identified naturally occurring splice variants of SAMHD1 , indicating that SAMHD1 expression and activity is regulated at a transcriptional level.
Interestingly, HIV-1 has no means of counteracting SAMHD1, and our recent study suggests that co-evolution of primate SAMHD1 and lentivirus Vpx led to the loss of the vpx gene in the HIV-1 precursor, SIVcpz, and consequently HIV-1 . Additional studies also suggested that SAMHD1 restriction toward HIV-1 was evolutionarily maintained under positive selection and that antagonism of SAMHD1 by Vpx is species-specific [51, 52], but that Vpx degradation of SAMHD1 was an acquired ability that arose through positive selection in lentiviruses . We recently reported that common polymorphisms of SAMHD1 are unlikely to contribute to the infection and natural control of HIV-1, at least in European and African-American individuals . It is interesting to investigate whether polymorphisms of SAMHD1 are associated with HIV-2 and SIV infections in humans and non-human primates, respectively.
SAMHD1 has been suggested to play a role in the innate immune responses to viral infections [54–58]. Our results indicate that SAMHD1 functions as an important restriction factor to counteract HIV-1 infection in DCs. Broader understanding of the mechanism of SAMHD1-mediated restriction in non-dividing cells and further investigation of the biological role of SAMHD1 is vital to enhancing our knowledge of HIV-1 infection and pathogenesis.
Our results suggest that SAMHD1-mediated reduction of the intracellular dNTP pool in DCs is a common mechanism of HIV-1 restriction in myeloid cells. Endogenous expression of SAMHD1 in primary DCs or CD4+ T-lymphocytes is not further upregulated by type I IFNs.
HIV-1 proviral vector pNL-Luc-E–R+ contains a firefly luciferase reporter gene was a kind gift from Dr. Nathaniel Landau (New York University School of Medicine) . HIV-1 proviral vector pNLAD8 (R5-tropic) was a kind gift from Dr. Eric Freed  (National Cancer Institute-Frederick). The SIV3+ plasmid, provided by Dr. Andrea Cimarelli , was used to produce SIVmac251 VLPs for delivery of Vpx into cells. A SIV3+ derivative, in which the vpx and vpr initiation codons were mutated, pSIVX-, was provided by Dr. Jacek Skowronski (Case Western Reserve School of Medicine) and has been described previously. Trans-complemented Vpx-containing VLPs were produced using pCG.myc.Vpx that expresses Vpx from HIV-2Rod, and human SAMHD1 expression plasmid pCG-F-HA-SAMHD1 (kind gifts from Dr. Jacek Skowronski) or empty vector controls were used for transfections and over-expression in cell lines.
Human PBMCs were isolated from the buffy coat of healthy blood donors as previously described . Primary CD14+ monocytes and CD4+ T-lymphocytes were isolated from PBMCs by positive selection as described previously [36, 62]. Immature DCs were generated from purified monocytes by treatment with granulocyte-macrophage colony-stimulating factor and IL-4 (50 ng/mL, R&D Systems) for 5 days as described previously [63, 64]. Primary resting CD4+ T cells were cultured in the presence of 20 IU/mL of recombinant IL-2 (obtained from the NIH AIDS Research and Reference Reagent Program) and activated by 5 μg/mL of PHA (Sigma-Aldrich) for 2 days (short-term activation) as previously described . DCs and CD4+ T-lymphocytes generated using these methods were more than 98% pure by flow cytometry analysis of surface markers as previously described [34, 36, 64]. Human embryonic kidney cell line HEK 293T cells, HeLa cells, and the HIV-1 indicator cell line GHOST/X4/R5 have been previously described [63, 64].
Generation of SIV VLP for DC transduction
SIV VLPs were generated by transfection of HEK293T cells with the appropriate plasmids, SIV3+ or SIV(X-), pCG.myc.Vpx and the VSV-G expressing vector pVSV-G as described . Two days post-transfection, supernatants were harvested and filtered through a 0.45 μM filter and layered over a 25-45% sucrose step gradient. Gradients were ultra-centrifuged at 28,000 × g for 90 min at 4°C. Supernatants were collected from the gradient interface, diluted in PBS and ultra-centrifuged through a 25% sucrose cushion at 28,000 × g for 90 min at 4°C. VLPs were recovered in medium by gentle rocking at 4°C for 3 hr, aliquoted and stored at −80°C (protocol adapted from ).
Treatment of DCs, CD4+T-lymphocytes and cell lines with type 1 IFNs
Cells were treated with a range of concentration of IFNs or mock treated with medium, as indicated and described previously [32, 34, 35]. At 24 hr post-treatment, lysates were harvested and analyzed by SDS-PAGE and immunoblotting. All IFNs and other cytokines were purchased from PeproTech.
Quantification PCR analysis of SAMHD1mRNA levels in IFN-treated DCs
Levels of SAMHD1 mRNA levels in DCs treated with 2000 U/mL IFNα, IFNβ, or IFNγ for 6 or 24 hr were quantified by SYBR-green real-time quantitative PCR analysis using primer sets and protocols previously described . Briefly, RNA from treated DCs at various time points was isolated using an RNeasy kit (QIAgen) and 200 ng of total RNA from IFN-treated DCs was used as input for cDNA synthesis, according to manufacturer’s instructions for SuperScript III First-Strand Synthesis System (Life Technologies).
Single-cycle, luciferase reporter HIV-1 stock (HIV-Luc/VSV-G) was generated by calcium phosphate co-transfection of HEK 293T cells with the pNL-Luc-E–R+ and pVSV-G as described . Replication-competent NLAD8 WT HIV-1 virus stocks were generated by transfection of HEK 293T cells with proviral vector pNLAD8 as described . All virus stocks were harvested 2 days post-transfection and filtered through a 0.45 μM filter. The infectious units of virus stocks were evaluated by limiting dilution on GHOST/X4/R5 cells as described . HIV-1 p24 concentrations of viral stocks were measured by ELISA (anti-p24-coated plates were purchased from the AIDS Vaccine Program, SAIC-Frederick, MD) as previously described .
HEK 293T cells were transfected using a calcium phosphate method to over-express SAMHD1 or vector controls, and cells were processed for downstream applications at 24 hr post-transfection. HeLa cells were transfected using the TransIT-HeLaMONSTER transfection kit (Mirus) according to the manufacturer’s instructions. At 24 hr post-transfection, cells were processed for HIV-1 infection assays, dNTP analysis, or cell lysates were harvested for immunoblot analysis.
Cells were harvested as indicated and lysed in cell lysis buffer (Cell Signaling) supplemented with protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were subjected to 12% SDS-PAGE and immunoblotting as described . Restore Western blot stripping buffer (Pierce) was used to strip antibodies from probed membranes. Super-signal chemiluminescence substrate (Pierce) was used to detect horseradish peroxidase-conjugated secondary antibodies. Polyclonal mouse antibody reactive to SAMHD1 (ab67820) was purchased from Abcam and used at 1 μg/mL in 5% milk in Tris-buffered-saline-Tween. Antibody to HLA-II was purchased from BD Biosciences. Tetherin antiserum was a kind gift from Dr. Paul Spearman (Emory University) and used as described previously  . Antibody specific to the HA-epitope (Ha.11 clone 16B12) was purchased from Covance. β-actin (Santa Cruz Biotechnologies) or GAPDH (AbD serotec) antibodies were used as loading controls. Immunoblotting images were captured using Molecular Imager ChemiDoc XRS instrument or Fujifilm Luminescent Image Analyzer (LAS 4000) and analyzed using Quantity One software (BioRad) or Multi Gauge V3.0 software (Fuji Film).
HIV-1 infection assays
HIV-1 infection assays using luciferase reporter viruses were performed using a multiplicity of infection (MOI) range of 0.5-2 as described previously [64, 67]. Cell lysates were obtained at the indicated times post-infection and analyzed for luciferase activity using a commercially available kit (Promega) according to the manufacturer’s instructions. Total cell protein was quantified using a BCA assay (Pierce) and all luciferase results were normalized to total protein content. For infection of DCs using replication-competent HIV-1, DCs (2.5×105) were transduced with Vpx-containing VLPs for 2 hr and then incubated with HIV-1NLAD8 (20 ng p24-equivalent, MOI of 0.5) for 2 hr as described [32, 34]. Cells were then washed thoroughly and cultured for the indicated times. Cell-free supernatants from the HIV-1-infected DCs were harvested for Gag p24 quantification by p24 ELISA at the indicated times post-infection. Whole cell lysates of HIV-1-infected DCs were subjected to immunoblotting for HIV-1 Gag detection as described .
Quantitative PCR analysis of HIV-1 cDNA
Levels of early and late reverse transcription products in infected DCs were quantified by SYBR-green based real-time quantitative PCR analysis using primer sets previously described [33, 34, 68]. Briefly, 100 ng of genomic DNA from HIV-1 infected DCs were used as input for the detection of early or late reverse transcription products. All virus stocks were treated with DNaseI (40 U/ml; Ambion) prior to infections to avoid plasmid DNA contamination. DNA from infected cells at various time points was isolated using a DNeasy Blood and Tissue kit (QIAgen).
Intracellular dNTP quantification of DCs
For dNTP analysis and quantification, cells were harvested and lysed in cold 65% aqueous methanol, heated to 95°C for 3 min and extracts dried in a speed vacuum. Reactions were prepared and analyzed as described previously . Briefly, extracts were incubated with 200 fmol substrate, 50 nM HIV-1 reverse transcriptase (RT), 10 μM oligonucleotide dT, and RT reaction buffer. Reactions were incubated for 5 min at 37°C and terminated using 10 μl 40 mM EDTA, 99% formamide and heated at 95°C for 5 min. For analysis, reaction products were separated on a 14% polyacrylamide-urea denaturing gel (SequaGel, National Diagnostics) and analyzed on a PhosphorImager (PerkinElmer). Product extension was quantified by densitometry (Quantity One) and dNTP content was back calculated from percent product extended. VLP + Vpx (24 hr) reactions were initially diluted 1:3 to prevent substrate from being fully extended. The calculation of intracellular dNTP concentrations (nM) was based on the reported volumes of DCs , HEK 293T cells , and HeLa cells .
Data were analyzed using a two-way ANOVA test and Student’s T-test and statistical significance was defined as p <0.05.
We thank the members of the Wu laboratory for helpful discussions. We thank Drs. Andrea Cimarelli, Eric Freed, Nathaniel Landau, Vineet KewalRamani, Jacek Skowronski, and Paul Spearman the kind gift of reagents. This work was supported in part by grant AI098524 and AI102822 to LW and grants GM104198 and AI049781 to BK, from the National Institutes of Health (NIH). LW is supported in part by the Public Health Preparedness for Infectious Diseases Program of The Ohio State University. The following reagents were obtained through the AIDS Research and Reference Reagent Program, National Institutes of Health: recombinant interleukin-2 from Dr. Maurice Gately, Hoffmann-La Roche Inc, and HIV-1 p24 monoclonal antibody (#24-2) from Dr. Michael Malim.
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