Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac

Abstract

Background: SAMHD1 is a nuclear protein that blocks lentiviral infection before reverse transcription in macrophages and dendritic cells. The viral accessory protein Vpx overcomes the SAMHD1-mediated lentiviral block by inducing its proteasomal degradation.

Results: Here, we identified the nuclear localization signal (NLS) of SAMHD1, and studied its contribution to restriction of HIV-1 and SIVmac. By studying the cellular distribution of different SAMHD1 variants, we mapped the nuclear localization of SAMHD1 to residues 11KRPR14. Mutagenesis of these residues changed the cellular distribution of SAMHD1 from the nucleus to the cytoplasm. SAMHD1 mutants that lost nuclear localization restricted HIV-1 and SIV as potently as the wild type protein. Interestingly, SAMHD1 mutants that localized to the cytoplasm were not degraded by nuclear Vpx alleles. Therefore, nuclear Vpx alleles require nuclear localization of SAMHD1 in order to induce its degradation. In agreement, SIVmac viruses encoding Vpx did not overcome the restriction imposed by the cytoplasmic variants of SAMHD1.

Conclusions: We mapped the NLS of SAMHD1 to residues 11KRPR14 and studied the contribution of SAMHD1 nuclear localization to restriction of HIV-1 and SIV. These experiments demonstrate that cytoplasmic variants of SAMHD1 potently block lentiviral infection and are resistant to Vpx-mediated degradation. The nuclear Vpx alleles studied here are only capable of degrading a nuclearly localized SAMHD1 suggesting that Vpx-mediated degradation of SAMHD1 is initiated in the nucleus.

Figure 1

The nuclear localization signal of SAMHD1 is contained in the first 150 amino acids. (A) Schematic representation of the different SAMHD1 GFP-fusion constructs. Full-length SAMHD1 is schematically represented, and the numbers of the amino acids residues at the boundaries of the protein domains are shown. The N terminus of the protein is fused to GFP, which is shown as its own structure. (B) HeLa cells expressing the indicated GFP-fusion constructs (green) were imaged by fluorescence microscopy. The cellular nuclei were stained by using DAPI (blue). Image quantification for three independent experiments is shown in Table 1. (C) Expression of SAMHD1 GFP-fusion constructs was analyzed by Western blotting using antibodies against GFP. The different constructs are indicated and the molecular weight markers are shown. Similar results were obtained in three independent experiments and a representative experiment is shown.

Figure 2

Identification of¹¹KRPR¹⁴as the functional SAMHD1 nuclear localization signal. (A) Diagram showing the amino acid substitutions in the putative nuclear localization signal of SAMHD1. (B) HeLa cells expressing the indicated GFP-SAMHD1 variant were imaged by fluorescence microscopy(green). The cellular nuclei were stained by using DAPI (blue). Image quantification for three independent experiments is shown in Table 1. (C) The expression of GFP-SAMHD1 variants in HeLa cells was measured by Western blotting using antibodies against GFP. (D) HeLa cells expressing the indicated SAMHD1-FLAG variants were fixed and immunostained using antibodies against FLAG (red). Similarly, cellular nuclei were stained by using DAPI(blue). Image quantification for three independent experiments is shown in Table 1. (E) Expression of the indicated SAMHD1-FLAG variants in HeLa cells was analyzed by Western blotting using antibodies against FLAG. As a loading control, cell lysates were Western blotted using antibodies against GAPDH. Similar results were obtained in three independent experiments and a representative experiment is shown.

Figure 3

The nuclear localization signal of SAMHD1 is transferable. (A) HeLa cells expressing a fusion between the NLS of SAMHD1 and the cytoplasmic GFP-muNS protein (KRPR-GFP-muNS) were imaged by fluorescence microscopy (green). As a control, the cytoplasmic distribution of the GFP-muNS protein is shown. Cellular nuclei were stained by using DAPI (blue). Image quantification for three independent experiments is shown in Table 1. (B) Similarly, HeLa cells expressing a fusion between the NLS of SAMHD1 and GFP (KRPR-GFP) were imaged by fluorescence microscopy(green). Cellular nuclei were stained by using DAPI (blue). Image quantification for three independent experiments is shown in Table 1. (C) The expression of the indicated fusion proteins was analyzed by Western blotting using antibodies against GFP. As loading controls, cell lysates were Western blotted using antibodies against GAPDH. Similar results were obtained in three independent experiments and a representative experiment is shown.

Figure 4

Contribution of SAMHD1 nuclear localization to restriction of HIV-1. Human monocytic U937 cells stably expressing the indicated mutant and wild type SAMHD1 proteins (A) were challenged with increasing amounts of HIV-1-GFP (B). As a control, U937 cells stably transduced with the empty vector LPCX were challenged with HIV-1-GFP. (C) U937 cells stably expressing the indicated SAMHD1-FLAG variants were fixed and immunostained using antibodies against SAMHD1 (green). The cellular nuclei were stained by using DAPI (blue). Mock represents wild type U937 cells. Image quantification for three independent experiments is shown in Table 1. (D) SAMHD1-silenced THP-1 cells were transiently transduced with the indicated wild type and mutant SAMHD1 proteins. Forty-eight hours post-transduction cells were differentiated and infected with HIV-1 LUC-G, which expresses luciferase as a reporter for infection. Luciferase activity was measured 24 hours post-infection. Results are expressed as fold increase luciferase activity in transduced over mock THP-1 cells. (E) The level of mutant and wild type SAMHD1 expression, in transduced SAMHD1-silenced THP-1 cells, was analyzed by Western blotting using anti-FLAG antibodies. As loading control, we analyzed cell extracts by Western blot using anti-tubulin antibodies. Similar results were obtained in three independent experiments and a representative experiment is shown.

Figure 5

Vpx_n-induced degradation of SAMHD1 requires nuclear localization of SAMHD1. (A) HeLa cells were cotransfected with plasmids allowing expression of SAMHD1-FLAG or SAMHD1-K11A-FLAG and HA-tagged Vpx from SIVmac 251(Vpx_mac251), SIVrcm-ng (Vpx_rcm-ng), HIV-2ROD (Vpx_ROD) or HIV-2B (Vpx_2B). Thirty-six hours post-transfection the cells were harvested, and the expression levels of SAMHD1 and Vpx were analyzed by Western blot using anti-FLAG and HA antibodies. As a loading control, cell extracts were Western blotted using antibodies against Tubulin. Similar results were obtained in three independent experiments and a representative experiment is shown. (B) Human monocytic U937 cells stably expressing the indicated mutant or wild type SAMHD1 proteins were challenged with SIVmac or SIVmacΔVpx -GFP reporter viruses. Infection was determined by measuring the percentage of GFP-positive cells. Fold of restriction was calculated by dividing the % of GFP-positive cells resulting from infecting U937 cells containing the empty vector pLPCX by the % GFP-positive cells resulting from infecting U937 stably expressing the indicated SAMHD1 variant. Similar results were obtained in three independent experiments and standard deviation is shown.