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. 2018 Jul 17;92(15):e00292-18.
doi: 10.1128/JVI.00292-18. Print 2018 Aug 1.

SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates Reactivation of Viral Latency in CD4 + T Cells

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Free PMC article

SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates Reactivation of Viral Latency in CD4 + T Cells

Jenna M Antonucci et al. J Virol. .
Free PMC article

Abstract

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in nondividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T cells, which are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 long terminal repeat (LTR) activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4+ T cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 LTR-driven gene expression at a transcriptional level. Tat coexpression abrogated SAMHD1-mediated suppression of HIV-1 LTR-driven luciferase expression. SAMHD1 overexpression also suppressed the LTR activity of human T-cell leukemia virus type 1 (HTLV-1), but not that of murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D R207N [HD/RN]) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not the T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR in vitro These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression potentially regulates viral latency in CD4+ T cells.IMPORTANCE A critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in nondividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4+ T cells. Furthermore, SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4+ T cells.

Keywords: HIV-1; LTR; SAMHD1; gene expression; latency; reactivation.

Figures

FIG 1
FIG 1
SAMHD1 suppresses HIV-1 LTR-driven luciferase expression. (A to E) An HIV-1 LTR-driven firefly luciferase (FF-Luc) construct was cotransfected with an empty vector (V) or increasing amounts of a plasmid encoding HA-tagged SAMHD1 (pSAMHD1) into HEK293T cells. Cotransfection of a construct encoding HSV TK-driven Renilla luciferase (Ren-Luc) was used as a control of transfection efficiency. (A) Overexpression of SAMHD1 was confirmed by immunoblotting. GAPDH was used as a loading control. Relative SAMHD1 expression levels were quantified by densitometry and normalized to GAPDH levels, with 1,000 ng of the pSAMHD1 sample set as 1. (B through E) Ren-Luc activity (B) and mRNA levels (C), and FF-Luc activity (D) and mRNA levels (E), were measured at 24 h posttransfection. (B) Ren-Luc activity was normalized to the total protein concentration. (D and E) FF-Luc activity and mRNA levels were normalized to Ren-Luc activity and mRNA levels, with vector levels set as 1. Error bars show standard deviations for at least three independent experiments as analyzed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (****, P ≤ 0.0001) from results for vector control cells. (F to H) FF-Luc and Ren-Luc constructs were expressed by nucleofection in THP-1 control (Ctrl) cells or SAMHD1 knockout (KO) cells. (F) SAMHD1 KO was confirmed by immunoblotting, with GAPDH used as a loading control. (G and H) Luciferase activity was measured at 48 h postnucleofection. Raw Ren-Luc values were normalized to total protein levels (G), and FF-Luc activity was normalized to Ren-Luc activity (H). Asterisks indicate significant differences (**, P ≤ 0.01) from results for control cells.
FIG 2
FIG 2
SAMHD1 suppresses gene expression driven by the LTR from HIV-1 or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven FF-Luc construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.
FIG 3
FIG 3
Nonphosphorylated and dNTPase-inactive SAMHD1 mutants show impaired suppression of HIV-1 LTR activity. (A to C) An HIV-1 LTR-driven FF-Luc construct was cotransfected with increasing amounts of plasmids encoding HA-tagged WT SAMHD1, the nonphosphorylated T592A mutant, or the dNTPase-inactive HD/RN mutant into HEK293T cells. Cotransfection of Ren-Luc was used as a control of transfection efficiency. (A) SAMHD1 expression was confirmed by immunoblotting. GAPDH was used as a loading control. Relative SAMHD1 expression levels quantified by densitometry were normalized to GAPDH levels. (B and C) Relative Ren-Luc units were normalized to the total protein concentration (B), and relative FF-Luc units were normalized to Ren-Luc levels (C). Vector cell luciferase activity was set as 1. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Error bars show standard deviations from at least three independent experiments. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector control cells.
FIG 4
FIG 4
WT SAMHD1 impairs HIV-1 reactivation in latently infected J-Lat cells. (A and C) HA-tagged WT SAMHD1, SAMHD1 mutants, or an empty vector (V) was stably expressed in J-Lat cells by lentiviral transduction. Relative SAMHD1 expression levels quantified by densitometry were normalized to GAPDH levels. (A and B) The cells were treated with either 10 ng/ml TNF-α or 32 nM PMA with 1 μM ionomycin (PMA+i). (A) At 24 h posttreatment, the expression of SAMHD1 was detected by immunoblotting, quantified by densitometry, and normalized to GAPDH levels. (B) The percentage of GFP-positive cells and the relative GFP mean fluorescence intensity (MFI) were determined by flow cytometry. (C) J-Lat cells expressing the T592A or HD/RN mutant were treated with 1× or 8× PMA+i (1× PMA+i corresponds to 16 nM PMA and 0.5 μM ionomycin), with expression of SAMHD1 measured and quantified by immunoblotting. (D) Latency reversal, as measured by the percentage of the cell population that was GFP positive and the MFI of GFP-positive cells, was determined by flow cytometry. Error bars in panels B and D represent standard deviations from at least three independent experiments analyzed by two-way ANOVA and Dunnett's multiple-comparison test. Asterisks indicate significant differences (**, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001) from results for vector cells in panels B and D.
FIG 5
FIG 5
WT SAMHD1 binds to HIV-1 proviral DNA in latently infected J-Lat cells. (A to D) J-Lat cells were seeded in the presence of PMA+i for 24 h. To achieve similar SAMHD1 expression levels, WT SAMHD1-expressing cells were treated with 4× PMA+i, while vector control cells and cells expressing T592A or HD/RN SAMHD1 were treated with 8× PMA+i. (A) SAMHD1 expression in input and IP lysates was analyzed by immunoblotting and densitometry. (B) Latency reversal, as measured by the percentage of the cell population that was GFP positive and the MFI of GFP-positive cells, was determined by flow cytometry. Three independent experiments were analyzed by two-way ANOVA and Dunnett's multiple-comparison test. Error bars represent the standard errors of the means. Asterisks indicate significant differences (***, P ≤ 0.001) from results for vector control cells. (C) Diagram of the locations of the qPCR amplicons. (D) Quantitative PCR data were normalized to spliced GAPDH levels and are presented as percentages of input in SAMHD1-expressing cells after normalization to vector background levels. Error bars represent the standard errors of the means from two independent experiments.
FIG 6
FIG 6
Specific binding of WT SAMHD1 to an HIV-1 LTR fragment in vitro. (A to C) Results of FA binding assays for WT, T592A mutant, or HD/RN mutant SAMHD1 binding to a 90-mer fragment of the HIV-1 LTR in 50 mM, 100 mM, or 150 mM monovalent ions (25, 50, or 75 mM [each] NaCl and KCl), respectively. (D) Binding to a 90-mer scrambled DNA oligonucleotide was also tested at 50 mM monovalent ions (25 mM [each] NaCl and KCl). Error bars indicate the standard deviations from three independent experiments.
FIG 7
FIG 7
Endogenous SAMHD1 impairs HIV-1 reactivation in latently infected primary CD4+ T cells. (A) Protocol summary. Naïve CD4+ T cells were isolated from PBMCs from three different healthy donors, activated by incubation with anti-CD3/CD28 antibody-coated beads, and infected with a single-cycle HIV-1 containing a GFP reporter pseudotyped with VSV-G (HIV-GFP) to produce a primary TCM model of latency. (B) GFP expression was measured in TCMs before HIV-GFP infection (day 0), as well as 2 and 10 days postinfection, by flow cytometry. SSC, side scatter. (C) After infection and culture to produce latently infected quiescent CD4+ T cells, the cells were transduced either with an empty vector or with lentiviral vectors containing SAMHD1-specific shRNA to knock down SAMHD1 expression. Endogenous SAMHD1 protein expression was measured at day 13 by immunoblotting, and GAPDH served as a loading control. (D and E) After stimulation of the cells with anti-CD3/CD28, T-cell activation was measured by surface staining of CD69 (D), and HIV-1 reactivation was measured by GFP expression by flow cytometry (E), at day 13. (E) Changes in the percentages and the MFI of GFP-positive cells from three donors are shown. *, P ≤ 0.05.

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