The ribonuclease activity of SAMHD1 is required for HIV-1 restriction

Abstract

The HIV-1 restriction factor SAM domain- and HD domain-containing protein 1 (SAMHD1) is proposed to inhibit HIV-1 replication by depleting the intracellular dNTP pool. However, phosphorylation of SAMHD1 regulates its ability to restrict HIV-1 without decreasing cellular dNTP levels, which is not consistent with a role for SAMHD1 dNTPase activity in HIV-1 restriction. Here, we show that SAMHD1 possesses RNase activity and that the RNase but not the dNTPase function is essential for HIV-1 restriction. By enzymatically characterizing Aicardi-Goutières syndrome (AGS)-associated SAMHD1 mutations and mutations in the allosteric dGTP-binding site of SAMHD1 for defects in RNase or dNTPase activity, we identify SAMHD1 point mutants that cause loss of one or both functions. The RNase-positive and dNTPase-negative SAMHD1D137N mutant is able to restrict HIV-1 infection, whereas the RNase-negative and dNTPase-positive SAMHD1Q548A mutant is defective for HIV-1 restriction. SAMHD1 associates with HIV-1 RNA and degrades it during the early phases of cell infection. SAMHD1 silencing in macrophages and CD4(+) T cells from healthy donors increases HIV-1 RNA stability, rendering the cells permissive for HIV-1 infection. Furthermore, phosphorylation of SAMHD1 at T592 negatively regulates its RNase activity in cells and impedes HIV-1 restriction. Our results reveal that the RNase activity of SAMHD1 is responsible for preventing HIV-1 infection by directly degrading the HIV-1 RNA.

Figure 1

The RNase but not the dNTPase function of SAMHD1 is required for HIV-1 restriction. (a) Purified GST-SAMHD1_WT (150 nM) was incubated for 30 min at 37 °C with 5′-end ³²P labeled nucleic acid substrates (20-mer, 50 nM). The RNA and DNA are indicated by red and blue lines, respectively. (b) An RNase activity assay performed for SAMHD1 mutants as described in Fig. 1a using 20-mer ssRNA substrates. (c) A dGTP-triphosphohydrolase assay was performed for GST-SAMHD1 variants as described previously. (d) The HA-tagged SAMHD1_WT and SAMHD1_Q548A proteins were purified from the PMA-differentiated U937 cells using HA-antibody. An RNase and a dGTP triphosphohydrolase assays were performed as described above. (e) Inhibition of the SAMHD1 RNase activity by dGTP. The assay was performed as in a in the presence of dGTP. (f) The intracellular dNTP pools were measured in U937 cells stably expressing SAMHD1 variants as described. (g) U937 cells stably expressing SAMHD1mutants were treated with PMA and infected by pLaiΔenvGFP3, a HIV-1-GFP reporter virus with the LAI backbone. After 48 h, the percentages of GFP-positive cells were analyzed using flow cytometry. The percentage of GFP-positive cells was calculated relative to the number of GFP-positive mock-transfected cells. In f and g, the data are presented as the mean ± s.d. of triplicate independent experiments. (* and ** indicate significant differences compared with the mock-transfected control at P < 0.05 and P < 0.001, respectively, using the two-tailed Student's t test).

Figure 2

SAMHD1 directly degrades HIV-1 RNA in human monocytic cells. (a) The total cellular RNA was extracted from SAMHD1-expressing, HIV-1-GFP-infected U937 cells. The HIV-1 RNA content was quantified by qRT-PCR using HIV-1 gag-specific primers. The data were normalized to an internal β-actin. (b) Mock- and SAMHD1_WT-expressing U937 cells were infected with HIV-1-GFP or HIV-1_D443N-GFP. The HIV-1 RNA content was quantified by qRT-PCR using HIV-1 gfp-specific primers. (c) Relative HIV-1 genome abundance in reads per kilobase per million mapped reads (RPKM) calculated from RNA-Seq data. (d) Coverage of the RNA-Seq data across the HIV-1 genome in U937 cells expressing SAMHD1_WT, SAMHD1_D207N and SAMHD1_D137N after HIV-1-GFP infection. Data are representative of two independent experiments (c,d). (e) shRNA-mediated silencing of SAMHD1 (left) or Vpx-mediated degradation of SAMHD1 (right) in THP-1 cells increases HIV-1 RNA stability. THP-1 cells expressing shRNAs, Vpx or negative control were treated with PMA for 24 h followed by infection with HIV-1-GFP. The total viral RNA was quantified by qRT-PCR at the indicated times post-infection. Knockdown efficiency was determined by immunoblot analysis. (f) Enrichment of HIV-1 genomic RNA in the SAMHD1 immunoprecipitates. U937 cells expressing HA-tagged SAMHD1 variants were infected for 90 min with HIV-1-GFP. The purified RNAs in the anti-HA immunoprecipitates were quantified by qRT-PCR using HIV-1 gag-specific primers. In a, b, e, and f, data are presented as the mean ± s.d. of triplicate independent experiments. (*P < 0.05 and **P < 0.001 versus Mock or shControl, two-tailed Student's t test).

Figure 3

SAMHD1 degrades HIV-1 RNA in primary human MDMs and CD4⁺ T cells. (a) CD14⁺ monocytes isolated from three donors were differentiated into MDMs. MDMs were transfected with siRNA to SAMHD1 or control siRNA and then infected with 100 ng of HIV-1-GFP. The efficiency of SAMHD1 knockdown was evaluated by qRT-PCR (left). Total viral RNA was quantified at the indicated times post-infection by qRT-PCR using gfp-specific primers. The data were normalized to an internal β-actin (middle). Quantification of GFP-positive cells by flow cytometry of MDMs (right). (b) Post-activated resting CD4⁺ T cells from three donors were transfected with siRNA to SAMHD1 or control siRNA and then infected with 100 ng of HIV-1-GFP. The efficiency of SAMHD1 knockdown (left), HIV-1 RNA levels (middle) and HIV-1 infectivity (right) were analyzed as in a. (c) Effect of Vpx treatment on the HIV-1 RNA stability in resting CD4⁺ T cells. Resting CD4⁺ T cells were challenged with HIV-1-GFP or HIV-1-GFP_D443N in the presence of Vpx VLP. HIV-1 RNA levels were quantified as in a. In a-c, data are presented as the mean ± s.d. of three experiments. (* and ** indicate significant differences compared with siControl or –Vpx VLP control at P < 0.05 and P < 0.001, respectively, using the two-tailed Student's t test).

Figure 4. Phosphorylation of SAMHD1 regulates RNase activity

(a–c) U937 cells stably expressing an empty vector (mock) or Flag-tagged SAMHD1 variants were treated with PMA and infected with 100 ng of pLaiΔenvGFP3. (a) Flow cytometric analysis 48 hpi to measure the percentage of GFP-positive cells. The percentage of GFP-positive cells was calculated relative to the number of GFP-positive mock-transfected cells (right). Western blot analysis of cell extracts using the anti–Flag antibody. GAPDH was used as a loading control. (b) HIV-1 genomic RNA content was quantified by qRT-PCR using HIV-1 gfp-specific primers. The data were normalized to an internal β-actin. (c) Recombinant SAMHD1_T592V and SAMHD1_T592D proteins were purified from E. coli. The RNase activity assay was performed as described in Fig. 1 using 5′-end ³²P labeled 20-mer ssRNA substrates. (d) Effect of SAMHD1 T592 phosphorylation on substrate binding. RIP analysis was performed as described in Fig. 2f, except that cell lysates were immunoprecipitated using an anti-Flag antibody. In a, b, and d, data are presented as the mean ± s.d. of triplicate experiments. (* and ** indicate significant differences compared with the mock-transfected control at P < 0.05 and P < 0.001, respectively, using the two-tailed Student's t test). e. Model for anti-HIV-1 activity mediated by SAMHD1 RNase function.