The Vpx lentiviral accessory protein targets SAMHD1 for degradation in the nucleus

Abstract

Sterile alpha motif domain- and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase that restricts the replication of lentiviruses in myeloid cells by hydrolyzing the cellular deoxynucleotide triphosphates to a level below that which is required for reverse transcription. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) encode the accessory protein viral protein X (Vpx) that counteracts SAMHD1. Vpx recruits SAMHD1 to a cullin4A-RING E3 ubiquitin ligase (CRL4), which targets the enzyme for proteasomal degradation. Vpx and SAMHD1 both localize to the nucleus of the cell. We identified the nuclear localization sequence (NLS) of SAMHD1 as a conserved KRPR sequence at amino acid residues 11 to 14. SAMHD1 lacking a functional NLS localized to the cytoplasm but retained its triphosphohydrolase and antiviral activities. However, cytoplasmic SAMHD1 was resistant to Vpx-induced degradation, and its antiviral activity was not counteracted by Vpx. Cytoplasmic SAMHD1 interacted with Vpx and retained it in the cytoplasm. The inhibition of nuclear export with leptomycin B did not impair the ability of Vpx to degrade SAMHD1. These findings suggest that SAMHD1 is targeted by Vpx for ubiquitination and degradation in the nucleus.

Fig 1

Identification of the SAMHD1 NLS. (A) The domain structure of SAMHD1 is shown at the top, and at the bottom is the ClustalW alignment of the first 36 amino acids in human (Homo sapiens), rhesus (Macaca mulatta), and mouse (Mus musculus) SAMHD1. A single NLS motif (boldface) was identified by analysis with cNLS mapper software (23). Identical residues are indicated by asterisks, strongly conserved residues by colons, weakly conserved residues by dots, and nonconservative changes by blank spaces. A ΔNLS mutant was generated in which the K11, R12, and R14 residues were changed to alanine. (B) The images show confocal immunofluorescence of HeLa cells transfected with HA-tagged human SAMHD1 (wild type [WT]), ΔNLS.SAMHD1 (ΔNLS), amino-terminal-deleted SAMHD1(residues 37 to 626), carboxy-terminal-truncated SAMHD1(residues 1 to 354), and mouse SAMHD1 isoform 2 (mu iso2) expression vectors. The transfected SAMHD1 was stained with an anti-HA antibody (red), and nuclei were counterstained with Hoechst (blue).

Fig 2

Cytoplasmic SAMHD1 retains dNTP hydrolase activity and restricts HIV-1 infection but is resistant to Vpx. (A) 293T cells were transfected with expression vectors for SAMHD1 (WT), catalytically inactive SAMHD1(H233A), cytoplasmic ΔNLS.SAMHD1 (ΔNLS), or an empty-vector control. The proteins were immunoprecipitated from cell lysates and incubated with [γ-³²P]ATP. The reaction products were separated by TLC and visualized by autoradiography. The immunoprecipitated proteins were visualized on an immunoblot using an anti-HA MAb. (B) Quantification of cellular dATP and dTTP pools in PMA-treated U937 cells expressing wild-type (WT) or mutant proteins was determined by the single-nucleotide incorporation assay (10). The SAMHD1(HD-AA) has alanine mutations in the HD204-205 residues within the HD domain. P values were generated using a paired one-tailed Student's t test: the single asterisk represents a P value of 0.007 (dATP) or 0.017 (dTTP), and the double asterisk represents a P value of 0.010 (dATP) or 0.019 (dTTP). (C) U937 cells expressing wild type (WT), mouse isoform 2 (mu iso2), or the indicated mutated SAMHD1 protein were differentiated with PMA and infected with an HIV.GFP reporter virus at multiplicities of infection (MOI) of 0.1, 0.3, and 1. GFP-positive cells were quantified by flow cytometry. (D) The SAMHD1 proteins expressed in the U937 cell lines are shown on an immunoblot probed with an anti-HA MAb. GAPDH was detected as a loading control. (E) PMA-differentiated clonal U937 cells expressing wild-type (WT) or ΔNLS.SAMHD1 (ΔNLS) or cells transduced with an empty vector (empty) were treated with Vpx-containing or control VLPs and then infected with an HIV.GFP reporter virus at MOI of 0.1, 0.3, and 1. GFP-positive cells were quantified by flow cytometry. P values were generated using a paired one-tailed Student's t test, where the single asterisk indicates a P value of 0.0006, two asterisks indicate a P value of 0.0004, and three asterisks indicate a P value of 0.0051.

Fig 3

Cytoplasmic SAMHD1 is resistant to Vpx- and Vpr-induced degradation. (A) U937 polyclonal cell populations stably expressing SAMHD1 (WT), ΔNLS.SAMHD1 (ΔNLS), and mouse isoform 2 (mu iso2) were differentiated with PMA. Vpx-containing or control VLPs were added. After 10 h, lysates were prepared, and SAMHD1 was visualized on an immunoblot probed with an anti-HA MAb. GAPDH was detected as a loading control. (B) Single-cell subclones derived from the polyclonal SAMHD1 cell lines that had similar levels of SAMHD1 were chosen. The susceptibility of the SAMHD1 proteins to Vpx-induced degradation was determined as described for panel A. (C) U937 polyclonal cell populations stably expressing SAMHD1 (WT) and ΔNLS.SAMHD1 (ΔNLS) were differentiated with PMA. Control VLPs or VLPs containing Vpx of SIVmac251 (VLP X⁺), SIVmac239, HIV-2 7312a, and SIVrcm or Vpr from SIVmus and SIVdeb were added for 10 h. The susceptibilities of the SAMHD1 proteins to degradation induced by the different Vpx and Vpr proteins were determined as described for panel A. SAMHD1 levels in cells treated with Vpx- or Vpr-containing VLPs were quantified relative to the cells treated with control VLPs (VLP X⁻). (D) The VLPs were pelleted by ultracentrifugation. They were solubilized, and the packaged Vpx or Vpr proteins were visualized on an immunoblot probed with an anti-Myc MAb (SIVmac239 Vpx) or an anti-FLAG MAb (Vpx from HIV-2 7312a and SIVrcm and Vpr from SIVmus and SIVdeb). SIV p27 was detected by probing with an anti-p27 MAb to confirm the presence of similar amounts of virions.

Fig 4

Vpx binds ΔNLS.SAMHD1 and is retained in the cytoplasm. (A) 293T cells were cotransfected with vectors that expressed Myc-tagged Vpx and HA-tagged wild-type or mutated SAMHD1. Two days posttransfection, the cells were incubated with MG132, and lysates were prepared. Vpx was immunoprecipitated with an anti-Myc MAb, and the complexes were analyzed on an immunoblot probed with anti-HA and anti-Myc MAbs. The cell lysates are shown in the upper lanes (input), and the immunoprecipitated (IP) complexes are shown in the lower lanes (myc-IP). (B) Confocal immunofluorescence analysis of HeLa cells stably expressing GFP-tagged SAMHD1 (WT) and ΔNLS.SAMHD1 (ΔNLS) transfected with an expression plasmid for Myc-tagged Vpx from SIVmac239. GFP-tagged SAMHD1 is shown in green, the transfected Vpx was stained with an anti-Myc antibody (red), and nuclei were counterstained with Hoechst (blue).

Fig 5

Vpx induces the degradation of SAMHD1 confined to the nucleus by LMB. (A) HeLa cell lines that stably expressed HA-tagged wild-type (WT) or ΔNLS/SAMHD1 (ΔNLS) were incubated with Vpx-containing or control VLPs in the presence or absence of leptomycin B (LMB). After 4 h, MG132 was added, and after an additional 6 h, cell lysates were prepared. SAMHD1 was visualized on an immunoblot probed with an anti-HA MAb and anti-GAPDH as a loading control. SAMHD1 levels in each sample were quantified relative to the level of SAMHD1 in cells treated with control VLPs (VLP X⁻) without the addition of LMB (−). (B) HeLa cells were transfected with pRev1.4-GFP-NES, a vector that expresses a Rev-GFP-NES fusion protein, and then treated for 3 h with 0, 1, and 10 ng/ml LMB. Rev-GFP-NES is shown in green, and the nuclei were stained with Hoechst (blue).