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The C-terminal domain of feline and bovine SAMHD1 proteins has a crucial role in lentiviral restriction

  • Chu Wang
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China

    The First Hospital and Institute of Immunology, Jilin University, Changchun 130012, China
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  • Kaikai Zhang
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China
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  • Lina Meng
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China
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  • Xin Zhang
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China
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  • Yanan Song
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China
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  • Ying Zhang
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China
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  • Yanxin Gai
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China
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  • Yuepeng Zhang
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China
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  • Bin Yu
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China

    Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China
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  • Jiaxin Wu
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China

    Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China
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  • Song Wang
    Affiliations
    Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130012, China
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  • Xianghui Yu
    Correspondence
    To whom correspondence should be addressed: Life Science Bldg., Jilin University, No. 2699 Qianjin St., Changchun 130012, Jilin Province, China. Tel.: 86-431-85167826
    Affiliations
    National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China

    Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China
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Open AccessPublished:February 19, 2020DOI:https://doi.org/10.1074/jbc.RA120.012767
      SAM and HD domain-containing protein 1 (SAMHD1) is a host factor that restricts reverse transcription of lentiviruses such as HIV in myeloid cells and resting T cells through its dNTP triphosphohydrolase (dNTPase) activity. Lentiviruses counteract this restriction by expressing the accessory protein Vpx or Vpr, which targets SAMHD1 for proteasomal degradation. SAMHD1 is conserved among mammals, and the feline and bovine SAMHD1 proteins (fSAM and bSAM) restrict lentiviruses by reducing cellular dNTP concentrations. However, the functional regions of fSAM and bSAM that are required for their biological functions are not well-characterized. Here, to establish alternative models to investigate SAMHD1 in vivo, we studied the restriction profile of fSAM and bSAM against different primate lentiviruses. We found that both fSAM and bSAM strongly restrict primate lentiviruses and that Vpx induces the proteasomal degradation of both fSAM and bSAM. Further investigation identified one and five amino acid sites in the C-terminal domain (CTD) of fSAM and bSAM, respectively, that are required for Vpx-mediated degradation. We also found that the CTD of bSAM is directly involved in mediating bSAM's antiviral activity by regulating dNTPase activity, whereas the CTD of fSAM is not. Our results suggest that the CTDs of fSAM and bSAM have important roles in their antiviral functions. These findings advance our understanding of the mechanism of fSAM- and bSAM-mediated viral restriction and might inform strategies for improving HIV animal models.

      Introduction

      Sterile alpha motif and histidine-aspartate-domain-containing protein 1 (SAMHD1) is an antiviral triphosphohydrolase that can reduce the concentration of intracellular deoxynucleoside triphosphates (dNTPs)
      The abbreviations used are: dNTP
      deoxynucleoside triphosphate
      SIV
      simian immunodeficiency virus
      SAM
      sterile alpha motif
      CTD
      C-terminal domain
      BIV
      bovine immunodeficiency virus
      FIV
      feline immunodeficiency virus
      MDBK
      Madin-Darby bovine kidney
      PMA
      phorbol 12-myristate 13-acetate.
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      • Mehrens J.
      • Wang C.
      • Gronenborn A.M.
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      HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.
      ,
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      ). Thus, the role of SAMHD1 in mice is unclear. By contrast, feline and bovine SAMHD1 (fSAM and bSAM) could restrict the lentiviruses of their own host species (feline and bovine immunodeficiency viruses, FIV and BIV, respectively) by using their dNTPase activities (
      • Mereby S.A.
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      Interplay of ancestral non-primate lentiviruses with the virus-restricting SAMHD1 proteins of their hosts.
      ). FIV and BIV also infect nondividing myeloid cells during their pathogenesis (
      • Brunner D.
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      Infection of peritoneal macrophages in vitro and in vivo with feline immunodeficiency virus.
      ,
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      ,
      • Bhatia S.
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      ) and can cause immune dysfunction like HIV (
      • Pedersen N.C.
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      Feline immunodeficiency virus infection.
      ,
      • Zhang S.
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      • Chen Q.
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      Immune suppression in calves with bovine immunodeficiency virus.
      ). Therefore, SAMHD1-knockout cats, and potentially cows, may be better models to study HIV pathogenesis and the biological function of SAMHD1, and the molecular mechanisms underlying feline and bovine SAMHD1-mediated restriction of HIV are worth being investigated.
      Here, we investigate the antiviral ability of fSAM and bSAM against different primate lentiviruses and the crucial regions in fSAM and bSAM that are required for their biological functions. We identified key sites in the C-terminal regions of fSAM and bSAM which are required for Vpx-mediated degradation and revealed the role of bovine SAMHD1 CTD in regulating the catalytic function and antiviral activity. Our findings will provide insights into the establishment of alternative models to investigate SAMHD1 in vivo.

      Results

      Antiviral activity of fSAM and bSAM against primate lentiviruses

      fSAM and bSAM are able to restrict FIV and BIV in SAMHD1-knockout THP-1 cells (
      • Mereby S.A.
      • Maehigashi T.
      • Holler J.M.
      • Kim D.H.
      • Schinazi R.F.
      • Kim B.
      Interplay of ancestral non-primate lentiviruses with the virus-restricting SAMHD1 proteins of their hosts.
      ). To facilitate the in vivo study of the anti-HIV activity of SAMHD1, we extended the analysis to their antiviral abilities against HIV-1, HIV-2, and SIV in vitro. We first constructed HA-tagged fSAM and bSAM expression vectors and tested their expression in TZM-bl cells. Human SAMHD1 (hSAM) was also expressed as a positive control, and cellular GAPDH protein was used as a loading control (Fig. 1A). Next, we developed a quick detection method to determine the antiviral activity of fSAM and bSAM by using TZM-bl cells. This cell line contains an HIV-1 Tat-regulated β-gal reporter gene that can be expressed after a single round of infection with not only HIV-1, but also HIV-2 and SIV (
      • Wei X.
      • Decker J.M.
      • Liu H.
      • Zhang Z.
      • Arani R.B.
      • Kilby J.M.
      • Saag M.S.
      • Wu X.
      • Shaw G.M.
      • Kappes J.C.
      Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.
      ). First, TZM-bl cells were transfected with human, feline or bovine SAMHD1 expression vector or empty vector. Next, the cells were detached at 48 h post transfection and re-seeded into 24-well plates with viral infection solutions containing HIV-1 (NL4–3), SIVmac239, or HIV-2 ROD. 48 h later, the infected cells were stained with the substrate of β-gal (X-Gal) and the number of positive blue cells was counted, by which the infectivity of the three viruses could be calculated. Consistent with previous findings (
      • Brandariz-Nuñez A.
      • Valle-Casuso J.C.
      • White T.E.
      • Laguette N.
      • Benkirane M.
      • Brojatsch J.
      • Diaz-Griffero F.
      Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac.
      ), hSAM showed strong antiviral activities against HIV-1, SIV, and HIV-2, which confirms the validity of this method (Fig. 1, B–D). fSAM and bSAM could also restrict the three viruses as efficiently as hSAM.
      Figure thumbnail gr1
      Figure 1The antiviral activity of feline and bovine SAMHD1 proteins against different primate lentiviruses. A, TZM-bl cells (2 × 106) were transfected with 3 μg pVR1012-homo-SAMHD1-HA, pVR1012-feline-SAMHD1-HA, or pVR1012-bovine-SAMHD1-HA or empty vector (pVR1012). 48 h post transfection, the transfected cells were detached with trypsin. Partial cells of equal amounts were harvested and the expression of human, feline, and bovine SAMHD1 proteins in the transfected cells was analyzed by Western blotting using anti-HA and anti-GAPDH antibodies. B–D, the remaining cells were re-seeded into 24-well plates (2 × 105 cells per well) with 300 μl viral infection solutions containing (B) HIV-1 NL4–3 (0.2 ng of RT), (C) SIVmac239 (1 ng of RT), or (D) HIV-2 ROD (1 ng of RT). 48 h later, the infected TZM-bl cells were stained with X-Gal and the positive blue cells were counted under optical microscope. Viral infectivity was determined by the number of blue cells. The viral infectivity in the empty vector–transfected cells was set to 100% (positive control, PC). Error bars represent the S.D. calculated from three independent infections. Statistical analysis was performed between the indicated groups using Student’s t test.
      To confirm this result, we generated hSAM-, fSAM-, and bSAM-expressing stable U937 cell lines which do not express endogenous SAMHD1 and stable U937 cell lines expressing catalytically inactive human, feline and bovine SAMHD1 proteins with alanine mutations in the HD domain (hSAMHD/AA, fSAMHD/AA, and bSAMHD/AA) (Fig. S1). After differentiated with PMA, the cells were infected with HIV-1, SIVmac239, or HIV-2 ROD. p24 antigen or reverse transcriptase (RT) levels in the culture supernatants were monitored for 5 days to determine the dynamic changes of viral replication. The results showed that hSAM, fSAM, and bSAM could significantly restrict the viral replication of these three viruses, whereas the catalytically inactive mutants could not (Fig. 2, A–C). Because Vpx expressed from SIVmac239 and HIV-2 ROD can induce the degradation of hSAM, the restriction efficiency of hSAM against SIVmac239 and HIV-2 ROD was lower than that against HIV-1 (Fig. 2, A–C). Similar phenomena were observed in fSAM and bSAM, indicating that Vpx may also mediate the degradation of these two SAMHD1 proteins. Immunoblot analysis of the U937 cells showed that the three pairs of WT SAMHD1 proteins and their catalytically inactive mutants were expressed at similar levels, respectively (Fig. 2D). We also constructed another catalytically inactive mutant for each of human, feline, and bovine WT SAMHD1 proteins (hSAMH233A, fSAMH233A, and bSAMH221A) (Fig. S1) and tested their antiviral activities against HIV-1, SIVmac239, and HIV-2 ROD using TZM-bl cells. As expected, the antiviral activity of these mutants was largely reduced (Fig. 2, E–G). We then tested the dNTPase activity of hSAM, fSAM, bSAM, and their catalytically inactive mutants in vitro by a malachite green–coupled dGTP-pyrophosphatase hydrolysis assay (
      • Seamon K.J.
      • Stivers J.T.
      A high-throughput enzyme-coupled assay for SAMHD1 dNTPase.
      ). In this assay, the final product inorganic phosphate (Pi) of the dGTP substrate hydrolysis by the SAMHD1 proteins was quantified to determine the dNTPase activity. The results confirmed that only few dGTP could be hydrolyzed by the catalytically inactive mutants (Fig. 2H). Together, these results demonstrated that feline and bovine SAMHD1 could restrict the infection and replication of primate lentiviruses by using their dNTPase activities.
      Figure thumbnail gr2
      Figure 2dNTPase activity-dependent restriction of viral replication mediated by feline and bovine SAMHD1 proteins in U937 and TZM-bl cells. A–C, empty U937 cells (5 × 105) or U937 cells stably expressing WT or catalytically inactive human, feline, or bovine SAMHD1 protein were differentiated with PMA for 20 h and infected with (A) HIV-1 NL4–3 (0.1 ng of RT), (B) SIVmac239 (1 ng of RT), or (C) HIV-2 ROD (1 ng of RT). The viral replication curves were measured by the concentration of p24 for HIV-1 or RT for SIV and HIV-2 in the culture supernatants collected on the day of infection (day 0) and at day 1, 3, and 5 post infection. Error bars represent the S.D. calculated from three independent infections. Statistical analysis was performed between each group of cells expressing WT SAMHD1 protein and the empty U937 cells using repeated-measure analysis of two-way ANOVA. D, the expression of SAMHD1 proteins in the U937 cell lines was analyzed by Western blotting using anti-HA and anti-GAPDH antibodies. E–G, TZM-bl cells (2 × 106) were transfected with 3 μg pVR1012-homo-SAMHD1-HA, pVR1012-feline-SAMHD1-HA, pVR1012-bovine-SAMHD1-HA, or their mutants or empty vector and then infected with (E) HIV-1 NL4–3 (0.2 ng of RT), (F) SIVmac239 (1 ng of RT), or (G) HIV-2 ROD (1 ng of RT) at 48 h post transfection. The viral infectivity in empty vector–transfected cells was set to 100% (PC). Error bars represent the S.D. calculated from three independent infections. Statistical analysis was performed between each group of the WT proteins and their mutants and between each mutant and PC using Student’s t test. H, in vitro detection of SAMHD1-catalyzed inorganic phosphate (Pi) release. HA-tagged SAMHD1 proteins were isolated from transfected HEK293 cells by immunoprecipitation. An aliquot of the immunoprecipitated SAMHD1 proteins was analyzed by Western blotting to ascertain comparable protein using anti-HA antibody. The levels of Pi released after in vitro dGTP-pyrophosphatase hydrolysis reactions were detected by malachite green. Error bars represent the S.D. calculated from three independent reactions.

      Proteosomal degradation of feline and bovine SAMHD1 by Vpx

      Vpx encoded by SIVmac and HIV-2 target the C-terminal domain of hSAM for proteosomal degradation to antagonize the antiviral function of hSAM (
      • Hrecka K.
      • Hao C.
      • Gierszewska M.
      • Swanson S.K.
      • Kesik-Brodacka M.
      • Srivastava S.
      • Florens L.
      • Washburn M.P.
      • Skowronski J.
      Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.
      ,
      • Laguette N.
      • Sobhian B.
      • Casartelli N.
      • Ringeard M.
      • Chable-Bessia C.
      • Ségéral E.
      • Yatim A.
      • Emiliani S.
      • Schwartz O.
      • Benkirane M.
      SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.
      ,
      • Ahn J.
      • Hao C.
      • Yan J.
      • DeLucia M.
      • Mehrens J.
      • Wang C.
      • Gronenborn A.M.
      • Skowronski J.
      HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.
      ,
      • Fregoso O.I.
      • Ahn J.
      • Wang C.
      • Mehrens J.
      • Skowronski J.
      • Emerman M.
      Evolutionary toggling of Vpx/Vpr specificity results in divergent recognition of the restriction factor SAMHD1.
      ). HIV-1 does not encode a Vpx protein, and its viral protein R (Vpr) does not counteract hSAM (
      • Lim E.S.
      • Fregoso O.I.
      • McCoy C.O.
      • Matsen F.A.
      • Malik H.S.
      • Emerman M.
      The ability of primate lentiviruses to degrade the monocyte restriction factor SAMHD1 preceded the birth of the viral accessory protein Vpx.
      ). It has been reported that FIV and BIV fail to proteosomally degrade the SAMHD1 proteins of their hosts (
      • Mereby S.A.
      • Maehigashi T.
      • Holler J.M.
      • Kim D.H.
      • Schinazi R.F.
      • Kim B.
      Interplay of ancestral non-primate lentiviruses with the virus-restricting SAMHD1 proteins of their hosts.
      ), and whether their genomes contain a certain accessory protein that functions as Vpx is unclear. To understand whether Vpx from primate lentivirus could mediate the degradation of fSAM and bSAM, we first examined the degradation of hSAM by SIVmac239 with or without Vpx as the control of degradation assay. HEK293 cells were co-transfected with the hSAM expression vector and SIVmac239 protein-expressing plasmid either with or without Vpx (pSIV or pSIVΔVpx). The expression levels of SAMHD1 and SIVmac239 were determined with anti-HA and anti-SIV p27 antibodies. As shown in Fig. 3A, WT SIVmac239 reduced the expression level of hSAM to 31.6% and the level was retrieved to 88.3% without the Vpx expression from SIVmac239 (lanes 3 and 4). Then, we tested the degradation of fSAM and bSAM by the WT or Vpx-deleted SIVmac239. Both of the proteins could be degraded by WT SIVmac239 but only about 15 and 30% of the total fSAM and bSAM were degraded (Fig. 3, B and C). To confirm the degradation result of fSAM, we repeated the immunoblot analysis in a feline kidney epithelial cell line, CRFK. We found that the degradation percentage of fSAM (about 45%) in the cell line of its own species was much higher than that in HEK293 cells (Fig. 3D). We also repeated the degradation assay of bSAM in Madin-Darby bovine kidney (MDBK) cells. However, because of the low transfection efficiency of this cell line, we did not obtain a clear and convincing result.
      Figure thumbnail gr3
      Figure 3Vpx induced degradation of feline and bovine SAMHD1 in a proteasome-dependent manner. A–C, HEK293 cells (1 × 106) were co-transfected with 600 ng of (A) pVR1012-homo-SAMHD1-HA, (B) pVR1012-feline-SAMHD1-HA, or (C) pVR1012-bovine-SAMHD1-HA and 1.5 μg of pSIVmac239 or pSIVmac239ΔVpx or empty vector. D, CRFK cells (1 × 106) were co-transfected with 600 ng of pVR1012-feline-SAMHD1-HA and 1.5 μg of pSIVmac239 or pSIVmac239ΔVpx or empty vector. Cells were harvested at 48 h post transfection and then analyzed by Western blotting using anti-HA, anti-SIV p27, and anti-GAPDH antibodies. The percentage of SAMHD1 in the presence of pSIVmac239 or pSIVmac239ΔVpx was calculated relative to that of the corresponding SAMHD1 in the absence of them (set to 100%). E–G, HEK293 cells (1 × 106) were co-transfected with 600 ng of (E) pVR1012-homo-SAMHD1-HA, (F) pVR1012-feline-SAMHD1-HA, or (G) pVR1012-bovine-SAMHD1-HA and 500 ng of pCG-Vpxmac-HA or empty vector. The transfected cells were treated with the proteasome inhibitor MG132 at 20 μm or DMSO as a negative control at 36 h after transfection. Cells were harvested 12 h later (48 h post transfection) and then analyzed by Western blotting using anti-HA and anti-GAPDH antibodies. The percentage of SAMHD1 in the presence of Vpxmac with DMSO or MG132 treatment was calculated relative to that of the corresponding SAMHD1 in the absence of Vpxmac (set to 100%). H, empty U937 cells (5 × 105) or U937 cells stably expressing each WT SAMHD1 protein were differentiated with PMA for 20 h and infected with SIVmac239 or SIVmac239ΔVpx (1 ng of RT). The viral replication curves were measured by the concentration of RT in the culture supernatants collected on the day of infection (day 0) and at day 1, 3, and 5 post infection. Error bars represent the S.D. calculated from three independent infections.
      Next, we investigated whether fSAM and bSAM were degraded via the proteosomal pathway. HA-tagged human, feline, or bovine SAMHD1 proteins were co-expressed with HA-tagged Vpx from the SIVmac239 (Vpxmac) or HIV-2 ROD (VpxROD) strain or empty vector in HEK293 cells in the presence of the proteasome inhibitor MG132 or DMSO as a negative control. The results showed that MG132 could block the Vpxmac- and VpxROD-induced degradation of fSAM and bSAM (Fig. 3, E–G and Fig. S2), indicating that fSAM and bSAM are both degraded proteasomally in HEK293 cells. We also infected the U937 cell lines stably expressing hSAM, fSAM, and bSAM with SIVmac239 and Vpx-deleted SIVmac239 (SIVΔVpx) viruses and quantified the RT levels in the culture supernatants for 5 days. The restriction efficiency of the three SAMHD1 proteins against Vpx-deleted SIVmac239 was higher than that against WT SIVmac239 (Fig. 3H), indicating that feline and bovine SAMHD1 were partially degraded by SIVmac239 Vpx in U937 cells. Collectively, these results demonstrated that Vpx is able to mediate the proteosomal degradation of feline and bovine SAMHD1, although this counteraction is weaker than that between Vpx and human SAMHD1.

      The function of fSAM and bSAM CTD related to Vpx-induced degradation

      Alignment of the C-terminal amino acid sequences of human, simian, feline, and bovine SAMHD1 proteins revealed sequence divergence in the CTD of fSAM and bSAM compared with that of primate SAMHD1 (Fig. 4A), especially in bSAM that lacks a typical C-terminal sequence. To determine how these domains mediate Vpx-induced degradation of fSAM and bSAM, we first constructed an hSAM variant truncated at the C terminus (hSAM1–600, truncated from amino acid 601 to 626) as a control. As shown previously (
      • Fregoso O.I.
      • Ahn J.
      • Wang C.
      • Mehrens J.
      • Skowronski J.
      • Emerman M.
      Evolutionary toggling of Vpx/Vpr specificity results in divergent recognition of the restriction factor SAMHD1.
      ), SIVmac239 Vpx was unable to degrade C-terminally truncated human SAMHD1 (Fig. 4B, lane 4). Next, we constructed a CTD-truncated fSAM variant (fSAM1–601, truncated from amino acid 602 to 627) and about 28% of fSAM in the HEK293 cells was retrieved (Fig. 4C, lane 4), suggesting that the CTD of fSAM contributed to the degradation induced by Vpx. To investigate whether hSAM CTD (hCTD, residue 601 to 626) containing the Vpx recognition sequence can increase the sensitivity of fSAM and bSAM to Vpx-mediated degradation, we then constructed a fSAM-hSAM chimeric protein by swapping the fSAM1–601 with hCTD (fSAM1–601+hCTD) and a bSAM-hSAM chimeric protein by adding the hCTD to the C-terminal end of bSAM (bSAM+hCTD). The co-transfection results in HEK293 cells demonstrated that the fSAM1–601+hCTD chimera was more sensitive to Vpx-mediated degradation than its WT SAMHD1 protein but the bSAM+hCTD chimera was not (Fig. 4C, lanes 6 and 10), indicating that bSAM has its own sequence other than the typical CTD sequence for mediating Vpx-induced degradation. To find this sequence, we constructed C-terminally truncated bSAM variants (bSAM1–572 and bSAM1–580, truncated off the last 17 and 9 amino acids, respectively) of which the region showing the lowest similarity to the SAMHD1 proteins from other species (Fig. 4A) was segmentally deleted. Compared with WT bSAM, the level of bSAM1–572 in HEK293 cells was about 31% higher in the presence of SIVmac239 Vpx, whereas no difference was observed between WT bSAM and bSAM1–580 (Fig. 4C, lanes 12 and 14), suggesting that the amino acid sequence from residue 573 to 580 is crucial to bSAM degradation mediated by Vpx.
      Figure thumbnail gr4
      Figure 4Vpx-induced degradation of the CTD mutants of feline and bovine SAMHD1. A, alignment of amino acids of the C-terminal region of human, simian, feline, and bovine SAMHD1 proteins. The residues in human SAMHD1 are numbered as the scale. Numbers with “f” or “b” prefix and arrows represent the position in feline or bovine SAMHD1 proteins. Mutations were made at the sites marked with red triangles and green diamonds in feline and bovine SAMHD1 sequences, respectively. B, C, F, G, HEK293 cells (1 × 106) were co-transfected with 600 ng of (B) pVR1012-homo-SAMHD1-HA or (C, F, G) pVR1012-feline-SAMHD1-HA or pVR1012-bovine-SAMHD1-HA or their indicated mutants and 500 ng pCG-Vpxmac-HA or empty vector. Cell lysates were analyzed by Western blotting with anti-HA and anti-GAPDH antibodies. The dashed lines in (G) indicate the border of two spliced blots. D and E, proposed models for the interface between (D) feline SAMHD1 CTD-Vpxmac-DCAF1 CTD and (E) bovine SAMHD1 CTD-Vpxmac-DCAF1 CTD. SAMHD1 is shown in red, Vpxmac and DCAF1 CTD are in blue and gray, respectively. Residues contributing to the interface are shown as sticks, hydrogen-bonding interactions as dashed lines. Interacting residues between SAMHD1 CTD and Vpxmac are highlighted by yellow, between SAMHD1 CTD and DCAF1 CTD by green.
      To identify which sites might be necessary for fSAM and bSAM CTD-Vpx binding and subsequent degradation, we performed computational homology modeling studies based on the hSAM CTD-SIVsm Vpx-DCAF1 CTD X-ray crystal structure (
      • Schwefel D.
      • Groom H.C.
      • Boucherit V.C.
      • Christodoulou E.
      • Walker P.A.
      • Stoye J.P.
      • Bishop K.N.
      • Taylor I.A.
      Structural basis of lentiviral subversion of a cellular protein degradation pathway.
      ) using Discovery Studio to predict the binding models of fSAM and bSAM CTD with SIVmac239 Vpx and human DCAF1 CTD. The modeling results showed one possible site in fSAM CTD interacting with Vpxmac, which is a hydrogen bond between Thr-619 of fSAM and Tyr-65 of Vpxmac helix 3 (Fig. 4D). For bSAM, although it lacks more than 20 amino acids at the C terminus compared with hSAM and fSAM (Fig. 4A), its C-terminal tail also forms a structure (two short helices and a loop in the middle) similar to that of hSAM (
      • Schwefel D.
      • Groom H.C.
      • Boucherit V.C.
      • Christodoulou E.
      • Walker P.A.
      • Stoye J.P.
      • Bishop K.N.
      • Taylor I.A.
      Structural basis of lentiviral subversion of a cellular protein degradation pathway.
      ) and fSAM (Fig. 4E). Lys-579 of bSAM was predicted to be hydrogen bonded to Tyr-65 of Vpxmac, and there is also a possible hydrogen bond between Arg-578 of bSAM and Asn-1090 of DCAF1. Therefore, we first tested whether fSAM with Thr-619 to alanine mutation (fSAMT619A, marked with red triangle in the sequence of fSAM in Fig. 4A) would impact its degradation mediated by Vpx. As expected, fSAMT619A was more resistant to SIVmac239 Vpx-induced degradation than its WT protein (Fig. 4F). Next, to further investigate which sites are crucial to Vpx-induced bSAM degradation, each amino acid except Pro-576 in the range of residues 573 to 580 was mutated to alanine individually (marked with green diamonds in Fig. 4A). We found that the bSAM mutants with Arg-578, Lys-579 or each of the three leucine (Leu-575, -577, and -580) mutations could resist SIVmac239 Vpx-mediated degradation in HEK293 cells (Fig. 4G, lanes 8, 10, 12, 14, and 16), indicating that these sites are required in the formation of bSAM-Vpx-CRL4DCAF1 E3 ubiquitin ligase complex. This is consistent with the homology modeling result and the hydrophobic side chains of Leu-575, Leu-577, and Leu-580 from bSAM form a hydrophobic interface between the N termini of Vpxmac helix 1 and 3 (Fig. 4E).
      Next, we tested whether the fSAM and bSAM mutants that could not be degraded in the presence of Vpx are not able to interact with Vpx. To do this, HA-tagged hSAM or hSAM1–600 were co-expressed with myc-tagged SIVmac239 Vpx or FLAG-tagged human DCAF1 in HEK293 cells to validate the further experiments by co-immunoprecipitation using anti-HA antibody-conjugated agarose beads (Fig. 5A). Previous studies have found that Vpx binds DCAF1 using conserved motifs in helix 1 and helix 3, which in turn recruits other components of the CRL4DCAF1 E3 ubiquitin ligase (
      • Srivastava S.
      • Swanson S.K.
      • Manel N.
      • Florens L.
      • Washburn M.P.
      • Skowronski J.
      Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection.
      ,
      • Bergamaschi A.
      • Ayinde D.
      • David A.
      • Le Rouzic E.
      • Morel M.
      • Collin G.
      • Descamps D.
      • Damond F.
      • Brun-Vezinet F.
      • Nisole S.
      • Margottin-Goguet F.
      • Pancino G.
      • Transy C.
      The human immunodeficiency virus type 2 Vpx protein usurps the CUL4A-DDB1 DCAF1 ubiquitin ligase to overcome a postentry block in macrophage infection.
      ,
      • Hrecka K.
      • Hao C.
      • Gierszewska M.
      • Swanson S.K.
      • Kesik-Brodacka M.
      • Srivastava S.
      • Florens L.
      • Washburn M.P.
      • Skowronski J.
      Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.
      ,
      • Laguette N.
      • Sobhian B.
      • Casartelli N.
      • Ringeard M.
      • Chable-Bessia C.
      • Ségéral E.
      • Yatim A.
      • Emiliani S.
      • Schwartz O.
      • Benkirane M.
      SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.
      ,
      • Ahn J.
      • Hao C.
      • Yan J.
      • DeLucia M.
      • Mehrens J.
      • Wang C.
      • Gronenborn A.M.
      • Skowronski J.
      HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.
      ) to facilitate hSAM ubiquitination and subsequent degradation through recognition of the C-terminal sequence of hSAM (
      • Ahn J.
      • Hao C.
      • Yan J.
      • DeLucia M.
      • Mehrens J.
      • Wang C.
      • Gronenborn A.M.
      • Skowronski J.
      HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.
      ,
      • Schwefel D.
      • Groom H.C.
      • Boucherit V.C.
      • Christodoulou E.
      • Walker P.A.
      • Stoye J.P.
      • Bishop K.N.
      • Taylor I.A.
      Structural basis of lentiviral subversion of a cellular protein degradation pathway.
      ,
      • Fregoso O.I.
      • Ahn J.
      • Wang C.
      • Mehrens J.
      • Skowronski J.
      • Emerman M.
      Evolutionary toggling of Vpx/Vpr specificity results in divergent recognition of the restriction factor SAMHD1.
      ), whereas hSAM alone was found to be unable to interact with DCAF1 in co-immunoprecipitation assays (
      • Ahn J.
      • Hao C.
      • Yan J.
      • DeLucia M.
      • Mehrens J.
      • Wang C.
      • Gronenborn A.M.
      • Skowronski J.
      HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.
      ,
      • Fregoso O.I.
      • Ahn J.
      • Wang C.
      • Mehrens J.
      • Skowronski J.
      • Emerman M.
      Evolutionary toggling of Vpx/Vpr specificity results in divergent recognition of the restriction factor SAMHD1.
      ). Consistent with the previous results, hSAM1–600 was unable to bind SIVmac239 Vpx (Fig. 5A, lane 6) and both hSAM and hSAM1–600 were unable to bind DCAF1 (lanes 2 and 5. No band of DCAF1 was detected in the co-immunoprecipitated complex, so the data were not shown.). DCAF1 could only be co-precipitated in the presence of Vpx (Fig. S3). The co-immunoprecipitation assays between DCAF1 and fSAM or bSAM showed that fSAM and bSAM also could not immunoprecipitate DCAF1 (Fig. 5B, lanes 2 and 6, the blank data of DCAF1 were not shown). Among the mutants of fSAM and bSAM, fSAMT619A and bSAML577A were unable to bind Vpx, and bSAML575A, bSAMK579A and bSAML580A demonstrated clearly decreased ability to interact with Vpx (Fig. 5B), confirming that mutations at these sites disrupted the interaction and hydrophobic interface of fSAM and bSAM with Vpx. Thus, it could be indicated that the abolished and decreased ability of Vpx to recruit these mutants to the E3 ubiquitin ligase complex leads to the inability of Vpx to degrade them.
      Figure thumbnail gr5
      Figure 5Essential amino acids of feline and bovine SAMHD1 proteins for Vpx binding. A and B, the interactions between (A) human SAMHD1 or (B) feline or bovine SAMHD1 or their indicated mutants and Vpxmac or DCAF1 were examined by co-immunoprecipitation in HEK293 cells. The transfected cells were incubated with 20 μm MG132 for 12 h before harvesting. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-HA, anti-FLAG, anti-myc, and anti-GAPDH antibodies.

      The impact of essential amino acids of fSAM and bSAM CTD on antiviral ability

      To test whether the mutations in the C terminus of fSAM and bSAM that compromised Vpx-induced degradation impact their antiviral activities, HIV-1, SIVmac239, and HIV-2 ROD were used to infect TZM-bl cells that were transfected with human, feline, or bovine WT SAMHD1 expression vector or each of their mutants or empty vector. 48 h later, the number of positive blue cells was counted to calculate the infectivity of the three viruses. Unlike WT hSAM, fSAM, and bSAM that could restrict all of the three viruses, respectively, we observed varied activity of each SAMHD1 mutant in restriction of the viruses (Fig. 6, A–C). hSAM1–600 and fSAM1–601 had no differences in restriction of the three viruses compared with their WT SAMHD1 proteins, but bSAM1–572 abolished the antiviral activity. fSAMT619A could slightly restrict the three viruses. bSAMK579A and bSAML580A also could restrict all of the three viruses but the antiviral ability of bSAMK579A was weakened, whereas bSAML575A and bSAMR578A only displayed mild antiviral activity against HIV-1 compared with WT bSAM. bSAMK577A could not restrict any of the three viruses. We also examined the expression level of all these SAMHD1 proteins in TZM-bl cells on the day of infection and found that the intracellular SAMHD1 levels were almost equal (Fig. 6D). These results demonstrated that CTD of hSAM and fSAM and Leu-580 of bSAM are not required for the antiviral function of these WT SAMHD1 proteins, and Thr-619 of fSAM and Leu-575, Lys-577, Arg-578 and Lys-579 of bSAM are partially or fully necessary for feline and bovine SAMHD1 in restriction of different viral strains.
      Figure thumbnail gr6
      Figure 6Restriction of different lentiviruses by feline and bovine SAMHD1 and their CTD mutants. A–C, TZM-bl cells (2 × 106) were transfected with 3 μg pVR1012-homo-SAMHD1-HA, pVR1012-feline-SAMHD1-HA, pVR1012-bovine-SAMHD1-HA, or their indicated mutants or empty vector and then 2 × 105 of the cells were infected with (A) HIV-1 (0.2 ng of RT), (B) SIVmac239 (1 ng of RT), or (C) HIV-2 ROD (1 ng of RT) at 48 h post transfection. The viral infectivity in empty vector-transfected cells was set to 100% (PC). Error bars represent the S.D. calculated from three independent infections. Statistical analysis was performed between each group and PC and between the indicated groups. D, the intracellular level of indicated SAMHD1 proteins in the transfected TZM-bl cells was analyzed by Western blotting using anti-HA and anti-GAPDH antibodies.
      To further understand why the antiviral activity of these CTD mutants were varied, we analyzed their dNTPase activities in vitro. The deletion of residues 601–626 of hSAM caused only a less than 2-fold reduction in the dNTPase activity (Fig. 7, A and D). Similarly, the deletion of fSAM CTD also caused a modest reduction in the dNTPase activity, and so did the T619A mutation of fSAM (Fig. 7, B and E). In contrast, the deletion of the CTD of bSAM largely impacted its dNTP hydrolysis function (Fig. 7, C and F). bSAML577A and bSAMR578A showed a more than 3-fold reduction in the dNTPase activity and bSAMR579A showed a half-reduced activity compared with the WT bSAM, whereas bSAML575A and bSAMR580A still had the same dNTP hydrolysis capacity as the WT bSAM (Fig. 7, C and F). Because significant differences in the dNTPase activity were observed between many mutants (hSAM1–600, fSAM1–601, fSAMT619A, bSAM1–572, bSAML577A, bSAMR578A, and bSAMK579A) and their WT SAMHD1 proteins but the antiviral activity of two of the mutants (hSAM1–600 and fSAM1–601) were not impacted (Fig. 6, A–C), we proposed that a 2-fold reduction in the dNTPase activity in vitro (Fig. 7, D–F) was the threshold of unaffected antiviral activity in our assays, suggesting that the antiviral activity of fSAM and bSAM CTD mutants largely depends on their dNTPase activity and the CTD of SAMHD1, especially of bovine SAMHD1, contributes to the maintenance of dNTPase activity of SAMHD1.
      Figure thumbnail gr7
      Figure 7In vitro dNTPase activity of feline and bovine SAMHD1 and their CTD mutants. A–C, in vitro detection of SAMHD1-catalyzed Pi release. HA-tagged SAMHD1 proteins were isolated from transfected HEK293 cells by immunoprecipitation. An aliquot of the immunoprecipitated SAMHD1 proteins was analyzed by Western blotting to ascertain comparable protein using anti-HA antibody. The levels of Pi released after in vitro dGTP-PPase hydrolyzation reaction were detected by malachite green. Error bars represent the S.D. calculated from three independent reactions. Statistical analysis was performed between each CTD mutant and its WT SAMHD1 protein. D–F, the reduction fold was calculated by dividing the average value of Pi concentration released from dGTP-PPase hydrolyzation reaction of the WT SAMHD1 protein by each of the triplicate values from each mutant. The threshold value of Pi concentration reduction fold is 2.

      Discussion

      Generation of SAMHD1-knockout animal models can facilitate the investigation of restriction of HIV-1 vectors and endogenous retroviruses in vivo and has been carried out in mice (
      • Wu L.
      SAMHD1 knockout mice: Modeling retrovirus restriction in vivo.
      ,
      • Rehwinkel J.
      Mouse knockout models for HIV-1 restriction factors.
      ). In the current study, we found that both fSAM and bSAM could restrict HIV-1, HIV-2, and SIV. In addition, they could be degraded via a proteasomal pathway mediated by Vpx, which is different from mouse SAMHD1 (mSAM) that cannot be degraded by Vpx because of the altered amino acid sequence in the CTD (
      • Ahn J.
      • Hao C.
      • Yan J.
      • DeLucia M.
      • Mehrens J.
      • Wang C.
      • Gronenborn A.M.
      • Skowronski J.
      HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.
      ). Because both fSAM and bSAM are able to restrict their endogenous retroviruses FIV and BIV, respectively, by reducing intracellular dNTP pool (
      • Mereby S.A.
      • Maehigashi T.
      • Holler J.M.
      • Kim D.H.
      • Schinazi R.F.
      • Kim B.
      Interplay of ancestral non-primate lentiviruses with the virus-restricting SAMHD1 proteins of their hosts.
      ), these findings indicate that the physiological functions of fSAM and bSAM in vivo might be similar to that of SAMHD1 in human. Therefore, these two species of mammals, especially cats, would be better models to study the in vivo function of SAMHD1. In fact, FIV-infected cat models have been widely used to study HIV pathogenesis (
      • Miller C.
      • Abdo Z.
      • Ericsson A.
      • Elder J.
      • VandeWoude S.
      Applications of the FIV model to study HIV pathogenesis.
      ). The BIV/cow model is also a good means for studying lentiviruses because there are no safety issues with BIV (
      • Bhatia S.
      • Patil S.S.
      • Sood R.
      Bovine immunodeficiency virus: A lentiviral infection.
      ). Thus, it is necessary to study the features of feline and bovine SAMHD1 to help with the establishment of the animal models.
      fSAM and bSAM were less sensitive to Vpx-mediated degradation than hSAM in HEK293 cells, however, more fSAM could be degraded in CRFK cells (Fig. 3, A–D), suggesting that Vpx from primate lentiviruses have evolved to counteract their host SAMHD1 proteins more efficiently and the feline E3 ubiquitin ligase complex may be more effective to its own targeted protein. Although overexpression of Vpx (Fig. 3, E–G), compared with co-transfection with SIVmac239 protein-expressing plasmid (Fig. 3, A–C), would result in more degradation of SAMHD1, our results clearly demonstrated that both fSAM and bSAM could be degraded in the presence of Vpx. This is not consistent with a previous study which found that Vpx from SIVmac239 did not mediate the degradation of feline and bovine SAMHD1 in HEK293T cells (
      • Mereby S.A.
      • Maehigashi T.
      • Holler J.M.
      • Kim D.H.
      • Schinazi R.F.
      • Kim B.
      Interplay of ancestral non-primate lentiviruses with the virus-restricting SAMHD1 proteins of their hosts.
      ). We are curious about the reason for the different results obtained by others and ourselves, so we repeated the degradation assay in HEK293T cells by co-expression of human, feline, or bovine SAMHD1 proteins with Vpxmac or VpxROD. Interestingly, fSAM and bSAM were almost not degraded in the presence of Vpxmac, and the degradation of hSAM was also weakened in our HEK293T cell line (Fig. S4A), whereas the levels of degradation of these three SAMHD1 proteins mediated by VpxROD were similar to what was observed in HEK293 cells (Figs. S2 and S4B). This suggested that in some circumstances, different cell lines may have different results, and the HEK293 cell line is more suitable for our investigations between Vpx and SAMHD1 in this study. By homology modeling based on the hSAM CTD-SIVsm Vpx-DCAF1 CTD X-ray crystal structure, two (Thr-619 and Ly-579) possible sites interacting with Vpxmac were predicted in fSAM and bSAM CTD, respectively, and Arg-578 of bSAM is a potential site interacting with DCAF1 (Fig. 4, D and E). All of these three sites were required for Vpx-induced degradation (Fig. 4, F and G), but only mutation at Thr-619 of fSAM and Lys-579 of bSAM abolished or decreased the interaction with Vpx (Fig. 5B). The interaction between Arg-578 of bSAM and DCAF1 could not be detected in our experiments because the WT bSAM was unable to interact with DCAF1, which is consistent with previous results obtained between human SAMHD1 and DCAF1 by co-immunoprecipitation (
      • Ahn J.
      • Hao C.
      • Yan J.
      • DeLucia M.
      • Mehrens J.
      • Wang C.
      • Gronenborn A.M.
      • Skowronski J.
      HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.
      ,
      • Fregoso O.I.
      • Ahn J.
      • Wang C.
      • Mehrens J.
      • Skowronski J.
      • Emerman M.
      Evolutionary toggling of Vpx/Vpr specificity results in divergent recognition of the restriction factor SAMHD1.
      ) although the X-ray crystal structure showed that Lys-622 of hSAM has a direct interaction with Asp-1092 of DCAF1 (
      • Schwefel D.
      • Groom H.C.
      • Boucherit V.C.
      • Christodoulou E.
      • Walker P.A.
      • Stoye J.P.
      • Bishop K.N.
      • Taylor I.A.
      Structural basis of lentiviral subversion of a cellular protein degradation pathway.
      ). Because our result showed that mutation at Arg-578 of bSAM did not impact the interaction between bSAM and Vpx but diminished Vpx-induced degradation, this site might be necessary to the stability of the structure of bSAM-Vpx-DCAF1 complex required for the subsequent degradation.
      Human SAMHD1 is highly expressed in HIV-1 nonpermissive cells such as THP-1, monocytes, and monocyte-derived dendritic cells, whereas it is absent from HIV-1–sensitive T cell lines such as Jurkat and SupT1, and myeloid cell line U937 (
      • Laguette N.
      • Sobhian B.
      • Casartelli N.
      • Ringeard M.
      • Chable-Bessia C.
      • Ségéral E.
      • Yatim A.
      • Emiliani S.
      • Schwartz O.
      • Benkirane M.
      SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.
      ). Among these cell lines, THP-1 and U937 are widely used in studying SAMHD1-mediated restriction and related events (
      • Lahouassa H.
      • Daddacha W.
      • Hofmann H.
      • Ayinde D.
      • Logue E.C.
      • Dragin L.
      • Bloch N.
      • Maudet C.
      • Bertrand M.
      • Gramberg T.
      • Pancino G.
      • Priet S.
      • Canard B.
      • Laguette N.
      • Benkirane M.
      • Transy C.
      • Landau N.R.
      • Kim B.
      • Margottin-Goguet F.
      SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates.
      ,
      • White T.E.
      • Brandariz-Nuñez A.
      • Valle-Casuso J.C.
      • Amie S.
      • Nguyen L.
      • Kim B.
      • Brojatsch J.
      • Diaz-Griffero F.
      Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1.
      ,
      • Laguette N.
      • Sobhian B.
      • Casartelli N.
      • Ringeard M.
      • Chable-Bessia C.
      • Ségéral E.
      • Yatim A.
      • Emiliani S.
      • Schwartz O.
      • Benkirane M.
      SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.
      ,
      • Brandariz-Nuñez A.
      • Valle-Casuso J.C.
      • White T.E.
      • Laguette N.
      • Benkirane M.
      • Brojatsch J.
      • Diaz-Griffero F.
      Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac.
      ,
      • Hofmann H.
      • Logue E.C.
      • Bloch N.
      • Daddacha W.
      • Polsky S.B.
      • Schultz M.L.
      • Kim B.
      • Landau N.R.
      The Vpx lentiviral accessory protein targets SAMHD1 for degradation in the nucleus.
      ,
      • Chen S.
      • Bonifati S.
      • Qin Z.
      • St. Gelais C.
      • Kodigepalli K.M.
      • Barrett B.S.
      • Kim S.H.
      • Antonucci J.M.
      • Ladner K.J.
      • Buzovetsky O.
      • Knecht K.M.
      • Xiong Y.
      • Yount J.S.
      • Guttridge D.C.
      • Santiago M.L.
      • Wu L.
      SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-κB and interferon pathways.
      ,
      • St. Gelais C.
      • Kim S.H.
      • Maksimova V.V.
      • Buzovetsky O.
      • Knecht K.M.
      • Shepard C.
      • Kim B.
      • Xiong Y.
      • Wu L.
      A cyclin-binding motif in human SAMHD1 is required for its HIV-1 restriction, dNTPase activity, tetramer formation, and efficient phosphorylation.
      ). Although TZM-bl is not a T cell line, this cell line was artificially modified with surface CD4 and CCR5 and is susceptible to infection by both R5 and X4 HIV-1 isolates (
      • Wei X.
      • Decker J.M.
      • Liu H.
      • Zhang Z.
      • Arani R.B.
      • Kilby J.M.
      • Saag M.S.
      • Wu X.
      • Shaw G.M.
      • Kappes J.C.
      Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.
      ). The widely adopted protocols for evaluating viral infectivity, 50% inhibitory concentration (IC50) of antiviral reagent and 50% inhibitory dose (ID50) of neutralizing antibody are built on this cell line (
      • Finnegan C.M.
      • Rawat S.S.
      • Puri A.
      • Wang J.M.
      • Ruscetti F.W.
      • Blumenthal R.
      Ceramide, a target for antiretroviral therapy.
      ,
      • Gao F.
      • Weaver E.A.
      • Lu Z.
      • Li Y.
      • Liao H.X.
      • Ma B.
      • Alam S.M.
      • Scearce R.M.
      • Sutherland L.L.
      • Yu J.S.
      • Decker J.M.
      • Shaw G.M.
      • Montefiori D.C.
      • Korber B.T.
      • Hahn B.H.
      • Haynes B.F.
      Antigenicity and immunogenicity of a synthetic human immunodeficiency virus type 1 group m consensus envelope glycoprotein.
      ,
      • Montefiori D.C.
      Evaluating neutralizing antibodies against HIV, SIV, and SHIV in luciferase reporter gene assays.
      ). Therefore, it would be convenient to use SAMHD1 transiently transfected TZM-bl cells to detect the antiviral activity of SAMHD1 in vitro. We obtained consistent results from TZM-bl and stable U937 cells for the antiviral ability of human, feline, and bovine SAMHD1 (Figure 1, Figure 2). Because SAMHD1 was overexpressed in these two cell lines before infection and the quantified amounts of viruses used for infection were relatively low in our experiments, the effect of degradation after SIV and HIV-2 infection should be limited and the antiviral activity of SAMHD1 was predominant to the results. Our results showed that the C-terminal residues 601 to 626 are not required for the antiviral activity of human SAMHD1 (Fig. 6, A–C). Consistent results were also found by Schwefel et al. (
      • Schwefel D.
      • Groom H.C.
      • Boucherit V.C.
      • Christodoulou E.
      • Walker P.A.
      • Stoye J.P.
      • Bishop K.N.
      • Taylor I.A.
      Structural basis of lentiviral subversion of a cellular protein degradation pathway.
      ). This result is supported by the finding that removal of residues 582 to 626 of hSAM had only a modest effect on the dNTPase activity and had essentially no effect on the capacity of hSAM to oligomerize (
      • Seamon K.J.
      • Bumpus N.N.
      • Stivers J.T.
      Single-stranded nucleic acids bind to the tetramer interface of SAMHD1 and prevent formation of the catalytic homotetramer.
      ). However, we found that deletion of the CTD of bSAM abolished its antiviral activity (Fig. 6, A–C), whereas the antiviral activity of CTD-deleted fSAM was not affected, suggesting that the function of CTD may be specific among different species. Consistent with this possibility, previous study found that CTD is important for the dNTPase activity of mSAM and this activity is regulated through tetramer stabilization by the CTD (
      • Bloch N.
      • Gläsker S.
      • Sitaram P.
      • Hofmann H.
      • Shepard C.N.
      • Schultz M.L.
      • Kim B.
      • Landau N.R.
      A highly active isoform of lentivirus restriction factor SAMHD1 in mouse.
      ). Mutations that disrupt the allosteric site or the tetramer interface of the protein prevent dNTPase activity (
      • Yan J.
      • Kaur S.
      • DeLucia M.
      • Hao C.
      • Mehrens J.
      • Wang C.
      • Golczak M.
      • Palczewski K.
      • Gronenborn A.M.
      • Ahn J.
      • Skowronski J.
      Tetramerization of SAMHD1 is required for biological activity and inhibition of HIV infection.
      ,
      • Bloch N.
      • Gläsker S.
      • Sitaram P.
      • Hofmann H.
      • Shepard C.N.
      • Schultz M.L.
      • Kim B.
      • Landau N.R.
      A highly active isoform of lentivirus restriction factor SAMHD1 in mouse.
      ). Thus, it is possible that the bSAM mutants altered the conformation of the C-terminal tail in such a way that they disrupted tetramerization, consequently inactivating the enzyme. The half-reduced dNTPase activity in vitro might reflect the threshold of intracellular dNTP levels for efficient viral transcription. These findings highlighted the role of CTD in regulating the catalytic function of bovine SAMHD1, and it will be important to investigate how these mutations influence the antiviral ability of fSAM and bSAM against FIV and BIV in future analysis.
      Taken together, our study demonstrated that SAMHD1 from the three species have many common features in antiviral ability against different viral strains and degradation pathway mediated by Vpx, but have distinct differences in the functional sites of CTD with regard to Vpx-induced degradation and antiviral activities. Thr-619 of fSAM was identified as one of the only two sites in the C-terminal region that were under positive selection during SAMHD1 evolution in the Carnivora clade, including cat, and was one of the five residues that directly contact Vpx in all mammals, whereas more than 10 sites were identified being under positive selection in the C-terminal region (from residue 586 to the end, based on the human sequence numbering) of SAMHD1 from the Cetartiodactyla clade, including cow (
      • Monit C.
      • Morris E.R.
      • Ruis C.
      • Szafran B.
      • Thiltgen G.
      • Tsai M.C.
      • Mitchison N.A.
      • Bishop K.N.
      • Stoye J.P.
      • Taylor I.A.
      • Fassati A.
      • Goldstein R.A.
      Positive selection in dNTPase SAMHD1 throughout mammalian evolution.
      ). This is consistent with our homology modeling results and suggests that bovine SAMHD1 may be under more intensive selection during evolution and that factors expressed by BIV or other viruses which target the CTD of bSAM may exist. The findings in this study will help to understand the SAMHD1 activities responsible for its viral restriction and will facilitate the improvement of SAMHD1/HIV animal models.

      Experimental procedures

      Cell culture

      HEK293T cells, HEK293 cells, TZM-bl cells, feline kidney CRFK cells, and MDBK cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). U937 cells were cultured in RPMI 1640 medium with 10% FBS and were differentiated using phorbol 12-myristate 13-acetate (PMA) (25 ng/ml) for 20 h before use. All cell lines were cultured at 37 °C and 5% CO2.

      Plasmids

      Plasmids pVR1012-homo-SAMHD1-HA, pCG-Vpxmac-HA, and pCG-DCAF1-FLAG were gifts from Dr. Wenyan Zhang. The DNA sequences coding for feline-SAMHD1 (GenBank accession no. XM_003983547.3) and bovine-SAMHD1 (GenBank accession no. NM_001075861.1) were amplified from feline and bovine genomic cDNA (reverse-transcripted from total mRNA isolated from CRFK and MDBK cells, respectively) by PCR, sequence confirmed, and inserted into a pVR1012 vector (
      • Hartikka J.
      • Sawdey M.
      • Cornefert-Jensen F.
      • Margalith M.
      • Barnhart K.
      • Nolasco M.
      • Vahlsing H.L.
      • Meek J.
      • Marquet M.
      • Hobart P.
      • Norman J.
      • Manthorpe M.
      An improved plasmid DNA expression vector for direct injection into skeletal muscle.
      ) at the XbaI and BamH I restriction sites. A HA tag was added to the C terminus of each SAMHD1 sequence. To generate the sequence-truncated, or point-mutated SAMHD1 fragments, pVR1012-homo-SAMHD1-HA, pVR1012-feline-SAMHD1-HA, and pVR1012-bovine-SAMHD1-HA were used as PCR templates. Chimeric fSAM1–601+hCTD and bSAM+hCTD fragments were constructed by overlapping PCR. All PCR products were digested and cloned into the XbaI and BamH I sites of pVR1012 vector with C-terminal HA tags. An myc tag was also added to the C terminus of SIVmac239 Vpx gene amplified from pCG-Vpxmac-HA for the co-immunoprecipitation assay. The PCR product was digested and cloned into the XbaI and BamH I sites of pVR1012 vector (pVR1012-Vpxmac-myc). Codon-optimized DNA sequence coding for HIV-2 ROD Vpx (UniProtKB accession no. P06939) were synthesized (GenScript, Piscataway, NJ) and inserted into the pVR1012 vector with a myc tag (pVR1012-VpxROD-myc). For generation of stable U937 cell lines, pLVX-puro vectors expressing SAMHD1 were constructed by inserting the HA-tagged SAMHD1 fragments into the EcoR I and BamH I restriction sites of pLVX-puro.

      Antibodies and chemicals

      Anti-HA mouse monoclonal antibody (mAb) (no. 901514) was obtained from Covance (Princeton, NJ). Anti-SIV p27 mAb was provided by the National Institutes of Health AIDS Reagent Program (NIH-ARP). Anti-FLAG mouse mAb (no. F1804) and the proteasome inhibitor MG132 in DMSO solution (no. M7449) were purchased from Sigma-Aldrich. Anti-myc mouse mAb (no. 05-419) was purchased from Millipore (Burlington, MA). Anti-GAPDH mouse mAb (no. 60004–1-Ig) was purchased from Proteintech (Rosemont, IL). Alkaline phosphatase-conjugated goat anti-mouse IgG was obtained from Jackson Immunoresearch (West Grove, PA).

      Western blotting

      DNA transfection was carried out using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Cells were harvested at 48 h after transfection, centrifuged at 3000 rpm for 5 min and lysed. The prepared protein samples were separated by electrophoresis in SDS-PAGE, followed by transferring onto nitrocellulose-membranes. After blocking in 5% nonfat milk, the membranes were probed with primary and secondary antibodies. Staining was carried out with 0.66% 5-bromo-4-chloro-3-indolyl phosphate and 0.33% nitro blue tetrazolium solutions in 0.1 m Tris-HCl, pH 9.5. Protein band intensities were determined by Adobe Photoshop CC 2017 software (San Jose, CA) and normalized by the level of GAPDH. The remaining percentage of SAMHD1 after degradation was calculated by dividing the normalized band intensity of SAMHD1 protein in the presence of Vpx by that of the corresponding SAMHD1 protein in the absence of Vpx.

      Viral infectivity assay

      Viral infectivity was measured by infecting TZM-bl indicator cells which contain an HIV-1 LTR promoter-β-gal expression cassette (
      • Wei X.
      • Decker J.M.
      • Liu H.
      • Zhang Z.
      • Arani R.B.
      • Kilby J.M.
      • Saag M.S.
      • Wu X.
      • Shaw G.M.
      • Kappes J.C.
      Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.
      ). Viruses were generated by transfection into HEK293T cells. The infectious molecular clones for generation of HIV-1 (NL4–3), SIVmac239, and HIV-2 ROD were provided by NIH-ARP. At 72 h post transfection, culture supernatants were harvested and centrifuged at 3000 rpm for 10 min at 4 °C to remove cell debris. The viral concentration was determined by measuring the concentration of RT using a Lenti RT Activity Kit (Cavidi, Uppsala, Sweden). TZM-bl cells transfected with or without SAMHD1 were detached with trypsin at 48 h post transfection and were re-seeded into 24-well plates (2 × 105 cells per well) in 300 μl viral infection solutions (containing HIV-1 of 0.2 ng RT, SIVmac239 of 1 ng RT or HIV-2 ROD of 1 ng RT, 40 nm diethylaminoethyl (DEAE)-dextran and DMEM). At 48 h after infection, the cells were fixed and stained with X-Gal, the substrate for β-gal, as described previously (
      • Wang C.
      • Jiang C.
      • Gao N.
      • Zhang K.
      • Liu D.
      • Wang W.
      • Cong Z.
      • Qin C.
      • Ganusov V.V.
      • Ferrari G.
      • LaBranche C.
      • Montefiori D.C.
      • Kong W.
      • Yu X.
      • Gao F.
      Immunologic and virologic mechanisms for partial protection from intravenous challenge by an integration-defective SIV vaccine.
      ). The viral infectivity was determined by the number of positive blue cells. Each experiment was performed in triplicate.

      Generation and infection of stable U937 cell lines

      Lentiviral stocks for overexpression of HA-tagged SAMHD1 mutants were generated by transfection of HEK293T cells with SAMHD1-expressing pLVX-puro vectors, psPAX2 packaging plasmid, and pVSV-G (Addgene, Watertown, MA). 72 h post transfection, lentiviral stocks were harvested, filtered, and used to infect U937 cells in the presence of DEAE-dextran (20 nm), after which cells were cultured and selected in RPMI 1640 with 1 μg/ml of puromycin as described previously (
      • Hofmann H.
      • Logue E.C.
      • Bloch N.
      • Daddacha W.
      • Polsky S.B.
      • Schultz M.L.
      • Kim B.
      • Landau N.R.
      The Vpx lentiviral accessory protein targets SAMHD1 for degradation in the nucleus.
      ). The expression of SAMHD1 in PMA-differentiated cells was confirmed by Western blotting. SAMHD1-expressing stable U937 cells (5 × 105) were differentiated with PMA for 20 h, infected with HIV-1 of 0.1 ng RT, SIVmac239 of 1 ng RT, SIVmac239ΔVpx of 1 ng RT or HIV-2 ROD of 1 ng RT for 4 h, and then washed three times with RPMI 1640 medium and cultured in 6-well plates with 2 ml of RPMI 1640 containing 10% FBS. The culture supernatant (200 μl) in each well was harvested regularly and the same volume of fresh medium was replenished. The viral replication was monitored by measuring the concentration of p24 (Alliance HIV-1 P24 ANTIGEN ELISA Kit, PerkinElmer) for HIV-1 or RT for SIV and HIV-2 in the culture supernatant. All infections were performed in triplicate.

      In vitro dNTP hydrolysis assay

      HEK293 cells in 12-well plates transfected with 600 ng HA-tagged SAMHD1 constructs were harvested at 48 h after transfection and washed twice with cold reaction buffer (pH 7.4, containing 50 mm Tris-HCl, 50 mm KCl, and 5 mm MgCl2), and then lysed in 250 μl lysis buffer (pH 7.4, containing 50 mm Tris-HCl, 150 mm NaCl, and 1% Triton X-100) supplemented with complete Mini Protease Inhibitor Mixture Tablets (Roche, South San Francisco, CA) at 4 °C for 40 min, followed by centrifugation at 16,000 × g for 15 min at 4 °C. Cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4 °C for 3 h. The samples were then washed three times with washing buffer (pH 7.4, containing 20 mm Tris-HCl, 100 mm NaCl and 0.05% Tween 20) and once with the reaction buffer. 40 μl of reaction buffer was then added to the bead pellet. 30% of the bead slurry was reserved as an input control, and the rest was diluted at a 1: 10 dilution with the reaction buffer and mixed with 1 mm dGTP and 0.01 unit pyrophosphatase (New England Biolabs, Ipswich, MA) to a final volume of 40 μl in triplicate reactions. The reactions were incubated at 37 °C for 2 h with occasional mixing and stopped by heating to 70 °C for 5 min. The reaction products were then diluted by 25-fold with reaction buffer and the Pi release was measured with a Malachite Green Detection Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

      Co-immunoprecipitation assay

      Cells in 6-well plates were harvested at 48 h after transfection, then washed twice with cold PBS and lysed in 250 μl lysis buffer supplemented with complete Mini Protease Inhibitor Mixture Tablets at 4 °C for 40 min. The cell lysates were centrifuged at 16,000 × g for 15 min and the supernatants were incubated with anti-HA beads at 4 °C for 3 h. Subsequently, the beads were washed three times with the washing buffer, resuspended with 30 μl 2× SDS loading buffer, boiled at 97 °C for 10 min, and then subjected to SDS-PAGE and immunoblotting.

      Homology modeling

      Homology modeling of target proteins was performed by using the protein modeling module of Discovery Studio 2.1 software package (Omaha, NE). The PDB file of the hSAM CTD-SIVsm Vpx-DCAF1 CTD X-ray crystal structure (ID: 4CC9) was used as a template. The original hSAM CTD and SIVsm Vpx sequences were aligned with the feline (residue 607 to 625) or bovine (residue 568 to 586) SAMHD1 CTD and SIVmac239 Vpx sequences. Modeling was performed at the medium optimization level with refined loop parameters, and no additional restraints were used. The best model of 10 models built was selected based on the quality estimation score and overall structure similarity. Because two (SAMHD1 and Vpx) of the three molecules in the crystal structure were remodeled, three modeling strategies: SAMHD1 first, Vpx first and both together, were tested independently. Generally, the interacting sites displayed in all three tests were further analyzed by Western blotting and co-immunoprecipitation. The images of remodeled structures were then modified by using PyMOL v1.8 (Schrödinger, LLC, New York, NY).

      Statistical analysis

      Data are shown as mean ± S.D. Significance is calculated by using unpaired Student’s t test or repeated-measure analysis of two-way ANOVA with PRISM v6 (GraphPad Software, Inc., La Jolla, CA). * indicates p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. ns indicates no significance.

      Author contributions

      C. W. and X. Y. conceptualization; C. W. and K. Z. data curation; C. W., K. Z., B. Y., J. W., and X. Y. formal analysis; C. W. and X. Y. funding acquisition; C. W., K. Z., and L. M. investigation; C. W. and K. Z. visualization; C. W. and X. Z. writing-original draft; L. M., X. Z., Y. S., Ying Zhang, Y. G., and Yuepeng Zhang methodology; S. W. software; X. Y. supervision; X. Y. writing-review and editing.

      Supplementary Material

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