Nuclear import of SAMHD1 is mediated by a classical karyopherin α/β1 dependent pathway and confers sensitivity to VpxMAC induced ubiquitination and proteasomal degradation
- Torsten Schaller1Email author,
- Darja Pollpeter†1,
- Luis Apolonia†1,
- Caroline Goujon1 and
- Michael H Malim1
© Schaller et al.; licensee BioMed Central Ltd. 2014
Received: 3 October 2013
Accepted: 27 March 2014
Published: 8 April 2014
The deoxynucleotide-triphosphate (dNTP) hydrolase sterile alpha motif domain and HD domain 1 (SAMHD1) is a nuclear protein that inhibits HIV-1 infection in myeloid cells as well as quiescent CD4 T-cells, by decreasing the intracellular dNTP concentration below a level that is required for efficient reverse transcription. The Vpx proteins of the SIVSMM/HIV-2 lineage of lentiviruses bind SAMHD1 and recruit an ubiquitin ligase, leading to polyubiquitination and proteasomal degradation.
Here, we have investigated the importance of nuclear localization for SAMHD1′s antiviral function as well as its sensitivity to the Vpx protein of SIVMAC. Using GST pull down assays, as well as RNA silencing approaches, we show that SAMHD1 preferentially uses karyopherin α2 (KPNA2) and a classical N-terminal nuclear localization signal (14KRPR17) to enter the nucleus. Reduction of karyopherin β1 (KPNB1) or KPNA2 by RNAi also led to cytoplasmic re-distribution of SAMHD1. Using primary human monocyte-derived macrophages (MDM), a cell type in which SAMHD1 is naturally expressed to high levels, we demonstrate that nuclear localization is not required for its antiviral activity. Cytoplasmic SAMHD1 still binds to VpxMAC, is efficiently polyubiquitinated, but is not degraded. We also find that VpxMAC-induced SAMHD1 degradation was partially reversed by ubiquitin carrying the K48R or K11R substitution mutations, suggesting involvement of K48 and K11 linkages in SAMHD1 polyubiquitination. Using ubiquitin K-R mutants also revealed differences in the ubiquitin linkages between wild type and cytoplasmic forms of SAMHD1, suggesting a potential association with the resistance of cytoplasmic SAMHD1 to VpxMAC induced degradation.
Our work extends published observations on SAMHD1 nuclear localization to a natural cell type for HIV-1 infection, identifies KPNA2/KPNB1 as cellular proteins important for SAMHD1 nuclear import, and indicates that components of the nuclear proteasomal degradation machinery are required for SAMHD1 degradation.
KeywordsSAMHD1 Nuclear import Karyopherin Vpx Macrophages HIV-1 NLS Ubiquitin linkage Innate immunity KPNA KPNB Impα Impβ
Host cells have evolved a number of antiviral factors that must be overcome by retroviruses to establish a productive infection. Intriguingly, early events in human immunodeficiency virus 1 (HIV-1) infection including uncoating, the liberation of the viral genome, as well as reverse transcription are vulnerable targets for restriction factors such as members of the APOBEC3 family, TRIM5α and TRIMCyp, and the recently identified sterile alpha motif (SAM) and HD domain containing 1 (SAMHD1), a dNTPase that inhibits HIV-1 in differentiated myeloid cells, as well as resting CD4+ T-cells [1–9]. SAMHD1 is targeted for proteasomal degradation by the lentiviral accessory proteins Vpx or Vpr, which recruit a cellular ubiquitin ligase complex. The activity of Vpx/Vpr from diverse primate immunodeficiency viruses is species-specific, and HIV-1 Vpr proteins do not counteract human SAMHD1 [10, 11]. As a result, HIV-1 infects cells of myeloid origin, such as dendritic cells or macrophages, with limited efficiency and addition of VpxMAC containing virus like-particles relieves this block . Intriguingly, HIV-1 infection of dendritic cells in the presence of Vpx causes dendritic cell activation and immune activation through an unknown sensor resulting in cytokine production, suggesting that infection of dendritic cells in vivo may be disadvantageous for sustained virus infection .
Mutations in SAMHD1 have been associated with Aicardi-Goutières syndrome (AGS) a condition presenting with increased type I interferon levels mimicking congenital viral infection [14, 15]. Wild type SAMHD1 is localized to the nucleus, while AGS causing mutations can disrupt nuclear localization leading to SAMHD1 accumulation in the cytoplasm [15, 16]. Recently, three independent groups have identified the nuclear localization signal (NLS) of human SAMHD1, and have demonstrated that disruption of this N-terminal motif results in cytoplasmic accumulation [17–19]. Hofmann et al. proposed that VpxMAC triggers SAMHD1 degradation specifically in the nucleus , while, in contrast, Laguette et al. proposed that nuclear export of SAMHD1 is required for its degradation by VpxMAC. In addition, Brandariz-Nuniz et al. suggested that VpxHIV-2/2B can degrade cytoplasmic SAMHD1 , which could not be confirmed by Hofmann et al. . The identification of determinants leading to resistance of cytoplasmic SAMHD1 to VpxMAC mediated degradation may therefore help to address these discrepancies.
The nuclear import of cargo is mediated through diverse pathways involving the action of karyopherins, a group of at least 20 proteins in humans . While karyopherin β (KPNB, importin β) family members can directly interact with some NLSs, they commonly engage their cargo indirectly through the recruitment of proteins of the karyopherin α (KPNA/importin α) family, of which there are at least 7 different members in human . Karyopherin α proteins can bind a diversity of NLSs including monopartite NLSs, consisting of a single cluster of basic amino acids, bipartite NLSs consisting of multiple clusters as well as additional nonclassical NLSs . The selection of nuclear import pathways for a particular cargo may vary, and specific NLS-KPNA interactions have been shown to be dependent on the cell type, as well as stages of cellular development or differentiation [23–27]. A recent report by Guo et al. employed co-immunoprecipitation experiments to investigate interaction between KPNB1 and SAMHD1, however systematic functional analyses of the importance of karyopherin α proteins or KPNB1 in SAMHD1 nuclear import have not been performed .
Here we have extended the characterization of SAMHD1 nuclear import requirements to primary monocyte-derived macrophages (MDM), a natural target cell for HIV-1, and have addressed the mechanism of resistance of cytoplasmic SAMHD1 to VpxMAC induced degradation. We have confirmed the NLS in human SAMHD1 and show that SAMHD1 is imported into the nucleus through a classical nuclear import pathway involving the cellular proteins karyopherin α2 (KPNA2) as well as karyopherin β1 (KPNB1). Depletion of either protein through RNAi results in a partial cytoplasmic redistribution of SAMHD1 and mutational inactivation of the NLS disrupts SAMHD1 binding to KPNA2. Consistent with observations made using cell lines, our data in MDM demonstrate that cytoplasmic SAMHD1 is still able to inhibit HIV-1 infection and is less sensitive to VpxMAC induced degradation. Interestingly, in MDM a substantial amount (~20%) of SAMHD1 NLS mutant localizes to the nucleus, suggesting either co-operative import with endogenous wild type SAMHD1 or the use of alternative import ways independent of the N-terminal NLS. We show that VpxMAC and VpxHIV-2/ROD both interact with SAMHD1 mutated in its NLS, and that wild type as well as NLS mutant SAMHD1 are targets for VpxMAC dependent mono- and polyubiquitination. These observations imply that the reduced sensitivity of cytoplasmic SAMHD1 to VpxMAC mediated degradation, as compared to nuclear SAMHD1, is not attributable to reduced binding of SAMHD1 to VpxMAC or by lower levels of SAMHD1 ubiquitination. Using specific ubiquitin lysine to arginine mutants we demonstrate differences in the ubiquitin linkage patterns between nuclear and cytoplasmic SAMHD1, suggesting a connection to the resistance of cytoplasmic SAMHD1 to VpxMAC induced degradation. These findings suggest that specific components of the nuclear proteasomal machinery are required for VpxMAC induced SAMHD1 degradation.
The N-terminus of SAMHD1 comprises a classical NLS
Karyopherin α members and karyopherin β1 (KPNB1) mediate SAMHD1 nuclear import
To verify KPNA2 binding to the identified N-terminal NLS in SAMHD1, we performed GST-pull down assays using GST-SAMHD1 or GST-SAMHD1K11A and HA-tagged KPNA2. We found that wild type SAMHD1 but not SAMHD1K11A interacted with HA-KPNA2 (Figure 2C, lane 5 vs. 6), suggesting that KPNA2 is recruited to the N-terminal NLS. We note that in cells expressing KPNA2 specific shRNAs, SAMHD1 was only partially redistributed to the cytoplasm, possibly pointing to a level of redundancy in usage of karyopherin α members. Performing GST pull down assays with HA-KPNA1, 3, 4, 5, or 6 suggested that KPNA1 and KPNA6 might also interact with SAMHD1. In both cases, however, substantial binding to GST-SAMHD1K11A was also seen (Additional file 1: Figure S3, lanes 23 and 28) implying either that detected binding represents background, or that karyopherin α interactions with other motifs within SAMHD1 may occur. Although it is possible that such interactions may underlie the low levels of SAMHD1K11A nuclear accumulation seen in some conditions (Figure 1E and F), their contributions to the overall nuclear import of SAMHD1 are likely to be of minor importance.
Karyopherin β1 (KPNB1) has been demonstrated to target KPNA2 bound cargo for nuclear import [30, 31]. We therefore investigated whether KPNB1 reduction would also lead to cytoplasmic re-distribution of SAMHD1 in HeLa cells. Knock-down of KPNB1 led to a substantial increase in the cytoplasmic mean fluorescence intensity (Figure 2B). Similar results were obtained in 293 T cells by western blot analysis detecting endogenous KPNB1, demonstrating moderate efficiency of KPNB1 reduction (Additional file 1: Figure S2B). Together these data suggest that KPNA2 is the predominant but possibly not the only karyopherin α that binds to the SAMHD1 NLS and that KPNB1 is also important for SAMHD1 nuclear import, indicating utilization of a classical KPNA/KPNB1 nuclear import pathway.
Cytoplasmic SAMHD1 is less sensitive to VpxMACinduced degradation
To exclude the possibility that residue K11 in SAMHD1 is a target site for ubiquitination and that substitution of this residue with alanine caused resistance to VpxMAC mediated degradation by disrupting a possible ubiquitnation target site, we analysed naturally occurring variations of the NLS in SAMHD1 sequences from other species for their impact on subcellular localization as well as sensitivity to VpxMAC induced degradation (Additional file 1: Figure S6A). We therefore replaced the KRPR motif in YFP-tagged human SAMHD1 with KRSR (Rattus norvegicus), KRLR (Chlorocebus pygerythrus, Erythrocebus patas), KRAR (Echinops telfairi, Monodelphis domestica, Gallus gallus, Macropus eugenii), KKCR (Pteropus vampyrus), NRPR (Erinaceus europaeus), KRLH (Chlorocebus tantalus), KRPG (Oryctolagus cuniculus) or KRAC (Anolis carolinensis). We first investigated the subcellular localization of the individual YFP-tagged SAMHD1 variants and found that four of the eight variations (NRPR, KRLH, KRPG and KRAC) were distributed mainly in the cytoplasm, whereas the other variants (KRSR, KRLR, KRAR and KKCR) were found predominantly in the nucleus (Additional file 1: Figure S6B).
Next, we investigated the sensitivity of these SAMHD1 chimerae to VpxMAC induced degradation. SAMHD1 NLS variants that localized predominantly to the cytoplasm (NRPR, KRLH, KRPG and KRAC) were mainly insensitive to VpxMAC mediated degradation, whereas variants that were found predominantly in the nucleus (KRSR, KRLR, KRAR and KKCR) were sensitive, further supporting the conclusion that nuclear localization of SAMHD1 is a prerequisite for VpxMAC initiated degradation (Additional file 1: Figure S6C). Our results suggest that K11 and R14 are critical for the nuclear import of human SAMHD1, and that sensitivity to VpxMAC induced degradation depends on SAMHD1 nuclear localization, as suggested by parallel studies [17, 18].
Cytoplasmic SAMHD1 retains antiviral activity against HIV-1
To extend our studies to a more relevant cell type in which SAMHD1 is naturally expressed, we investigated cytoplasmic SAMHD1 sensitivity to VpxMAC in human MDM. Cells were isolated from 2 independent donors, transduced with lentiviral vectors encoding HA-tagged wild type or SAMHD1K11A, or E2-Crimson as a control, in the presence of VLPs with or without VpxMAC. Three days post transduction, the cells were infected with a HIV-1 GFP reporter vector and analyzed by flow cytometry. At the time of challenge, parallel samples were subjected to western blot or immunofluorescence analysis.
Since the presence of VpxMAC renders MDM more susceptible to lentiviral transduction, we first compared transduction efficiencies in the presence or absence of VpxMAC. We observed 10% of cells expressing E2-Crimson in the absence of VpxMAC and almost 100% in the presence of VpxMAC, confirming the functionality of VpxMAC (Additional file 1: Figure S8A). In parallel samples we investigated the levels of ectopic wild type or SAMHD1K11A, as well as the endogenous protein. Similar levels of HA-SAMHD1 and HA-SAMHD1K11A were detected in the absence of VpxMAC (Figure 5C, lane 3 and 5), suggesting comparable transduction efficiencies for both lentiviral vector stocks (Figure 5C and Additional file 1: Figure S8B, C). In the presence of VLPs containing VpxMAC, the level of HA-SAMHD1 was slightly increased (Figure 5C and Additional file 1: Figure S8B, C, lane 3 and 4), whereas the level of HA-SAMHD1K11A was substantially increased (Figure 5C and Additional file 1: Figure S8B, C, lane 5 and 6). We conclude that cytoplasmic SAMHD1 is less sensitive to VpxMAC initiated degradation in primary MDM than its nuclear localized counterpart. We also investigated the antiviral phenotypes of these cultures and observed a ~2-fold reduction in HIV-1 GFP reporter vector infectivity for HA-SAMHD1, but a substantially stronger reduction of ~5-10-fold in HA-SAMHD1K11A expressing cells (Figure 5D and Additional file 1: Figure S8D, E).
Our results indicate that cytoplasmic SAMHD1K11A can reduce HIV-1 infection in primary MDM and is substantially less sensitive to VpxMAC mediated suppression relative to nuclear localised SAMHD1. They also imply that SAMHD1K11A can be imported into the nucleus in MDM either by cooperative nuclear import in complex with endogenous SAMHD1 , or by alternative nuclear import pathways that are independent of the N-terminal NLS .
VpxMACinduces ubiquitination of nuclear and cytoplasmic SAMHD1
SAMHD1K11A has reduced sensitivity to VpxMAC stimulated degradation, but the underlying mechanism has not been defined. The CUL4A/DDB1/DCAF1 ligase complex is found in the nucleus and we speculated that the insensitivity of SAMHD1K11A may be due to the absence or unavailability of the relevant ubiquitination machinery in the cytoplasm.
To address the incorporation of other ubiquitin linkages in polyubiquitinated chains we co-transfected HA-SAMHD1WT or HA-SAMHD1K11A expressing plasmids with Flag-VpxMAC or Flag-VpxRCM and HIS-tagged wild type ubiquitin or ubiquitin lysine to arginine mutants UbiK6R, UbiK11R, UbiK27R, UbiK29R, UbiK33R, UbiK48R or UbiK63R and performed NiNTA pull down experiments as before. Interestingly, in addition to the enhanced accumulation of chains in the presence of UbiK48R, we also observed higher levels of polyubiquitin chains in the presence of UbiK11R (Figure 7B, lane 5 vs. 7), suggesting that K11 linked ubiquitin chains may also form during VpxMAC induced degradation of SAMHD1WT. We also observed a very strong accumulation of higher molecular mass bands with SAMHD1K11A in the presence of UbiK11R and a moderate increase in the presence of UbiK6R, indicative of perturbed deubiquitination in the presence of either of these mutants (Figure 7B, lane 7 and 8) as well as with K48R.
Here we have investigated the nuclear localization requirements for SAMHD1 and the consequences of its disruption. In cells from healthy human donors, SAMHD1 appears to be localized exclusively to the nucleus, whereas in cells of Aicardi-Goutières syndrome (AGS) patients with mutations in SAMHD1 cytoplasmic accumulation of SAMHD1 has been observed [15, 16]. We have identified a classical/basic NLS in the N-terminus of SAMHD1 (11KRPR14) and have shown that substitutions of critical amino acids in this motif result in cytoplasmic accumulation in MDM (Figure 1E). Consistent with recently published studies, NLS function can be transferred to a heterologous protein (Figure 1C)  and the SAMHD1 NLS can be substituted by a heterologous NLS (Figure 1D). Importantly, ~80-95% of the SAMHD1K11A NLS-mutant protein was localized to the cytoplasm in both HeLa cells as well as MDM, suggesting that the N-terminal NLS constitutes the major signal for nuclear import in cell lines as well as primary cells.
Systematic analysis of human KPNAs identified KPNA2 as the principal nuclear import receptor binding to the N-terminal SAMHD1 NLS (Figure 2), though a minor, possibly redundant, role for KPNA6 could not be ruled out (Additional file 1: Figure S3). We further confirmed utilization of the classical nuclear import pathway by demonstrating the importance of KPNB1 using RNAi (Figure 2). These data therefore indicate that human SAMHD1 nuclear import is primarily mediated through the interaction of KPNA2 with the N-terminal 11KRPR14 motif and engagement of KPNB1. Of note, two unrelated proteins, NBS1 and Oct4, contain almost identical NLSs only differing in the third amino acid position, 465KRER468 and 61KRKR64, respectively and both have been demonstrated to bind and require KPNA2 for nuclear import [37–40]. We did not observe a complete block for SAMHD1 nuclear import in cells treated with KPNA2 or KPNB1 specific shRNAs, which may be explained by cytotoxicity when KPNA2 or KPNB1 levels are largely ablated, or may reflect a degree of redundancy between nuclear import receptors. More specifically, we observed that KPNA1 interacts with both SAMHD1WT as well as SAMHD1K11A suggesting that SAMHD1 contains a KPNA1 binding site that is discrete from the N-terminal NLS. Although karyopherin α proteins other than KPNA2 may contribute to SAMHD1 nuclear import, it is likely that any such effects are of minor importance given that mutation of the N-terminal NLS is sufficient to re-distribute 80-95% of total SAMHD1 to the cytoplasm. However, we found that in MDM SAMHD1K11A was not completely excluded from the nucleus (Figure 1). The modest nuclear accumulation of SAMHD1K11A in MDM may be due to such activities, or assisted import as a complex with endogenous SAMHD1, as has been suggested for other proteins  and is supported by the data of Guo et al. .
Surprisingly, substitution of the NLS in human SAMHD1 with corresponding sequences from certain species caused cytoplasmic accumulation, suggesting that the NLS is not highly conserved between different species. Interestingly, African green monkeys contain two different SAMHD1 NLS sequences  [KRLH (Chlorocebus tantalus) and KRLR (Chlorocebus pygerythrus)] leading to cytoplasmic or nuclear localization of SAMHD1, respectively (Additional file 1: Figure S6). The physiological relevance of these differences is not yet known but may help illuminate the impact of SAMHD1 mislocalization on AGS . Importantly, we investigated the antiviral activity of cytoplasmic and nuclear SAMHD1 in MDM and confirmed that each form inhibited HIV-1 infection to a comparable degree (Figure 5) [17, 18]. This suggests that if mislocalization of mutated SAMHD1 contributes to the development of AGS, then SAMHD1′s HIV-1 restriction activity may be mechanistically distinct from its physiologic function in human cells.
Cytoplasmic SAMHD1 is less sensitive to VpxMAC mediated degradation in MDM (Figure 6), in keeping with what has been shown in cell lines [17, 18]. The mechanistic basis for the reduced sensitivity of cytoplasmic SAMHD1 to VpxMAC induced degradation can not be explained by reduced VpxMAC binding (Figure 3) . We therefore examined whether cytoplasmic SAMHD1 is ubiquitinated in response to VpxMAC. The degradation of nuclear (wild type) SAMHD1 in the presence of VpxMAC was partially prevented by the presence of dominant negative ubiquitin mutants K48R or K11R, suggesting that K48- as well as K11-linked polyubiquitination may precede SAMHD1 proteasomal degradation (Figure 7). Cytoplasmic SAMHD1K11A was also targeted for ubiquitination by VpxMAC (Figure 7), and our data invoke (at least) two plausible explanations for the reduced sensitivity of cytoplasmic SAMHD1 to VpxMAC induced degradation: 1) wild type SAMHD1 may be degraded using proteasomal machinery components such as deubiquitinating enzymes (DUBs) that are either confined to the nucleus, or only active in the nucleus; and/or 2) ubiquitin chains that are appended to cytoplasmic SAMHD1 may differ in the linkage patterns, leading to resistance to degradation. Using ubiquitin lysine to arginine mutants we observed differences in the polyubiquitination profiles for wild type and K11A SAMHD1, specifically with UbiK11R and UbiK6R, leading to increases in polyubiquitinated SAMHD1K11A compared to SAMHD1WT (Figure 7B, lanes 7 and 8). Importantly, DUBs that specifically modulate ubiquitination in the nucleus have been described before [41, 42] and future investigations of SAMHD1 will explore these possible scenarios with a view to providing insight into the nucleocytoplasmic partitioning of elements of the ubiquitin/proteasome system.
Our work extends published observations on SAMHD1 nuclear localization to a natural cell type for HIV-1 infection, identifies KPNA2/KPNB1 as cellular factors important for SAMHD1 nuclear import, and indicates that components of the nuclear proteasomal degradation machinery are required for Vpx induced SAMHD1 degradation.
Cell culture and stable cell lines
293 T, HEK-Blue™ (Invivogen) and Hela cells were grown in Dulbecco’s modified Eagle medium (DMEM); U937, THP-1 and primary monocyte-derived macrophages (MDM) were grown in RPMI 1640 Glutamax + HEPES medium supplemented with 10% fetal bovine serum (FBS) (Gibco) and penicillin-streptomycin. MDM were obtained from peripheral blood mononuclear cells (PBMCs) of healthy volunteer donors. Briefly, PBMCs were isolated using Lymphoprep (Axis-Shield) and monocytes were obtained by positive selection using CD14 specific MicroBeads (Miltenyi Biotec). After 3 h in serum-free RPMI 1640 medium, monocytes were differentiated into macrophages by culture for 4 to 10 days in growth media supplemented with 100 ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF) (R&D Systems).
Plasmids and shRNAs
U937 cells were transduced with the HIV-1 based lentiviral vector, pCSCW, where the human SAMHD1 cDNA had replaced the mCherry open reading frame to express untagged wild type or K11A mutant SAMHD1, pCSwtW and pCSk11aW, respectively. For ectopic expression in monocyte-derived macrophages (MDM) a HA-tag coding sequence was inserted 5′ of the SAMHD1 open reading frame generating pCSHAwtW and pCSHAk11aW. To generate a control that was suitable for measuring MDM transduction efficiency by flow cytometry the E2-Crimson cDNA was inserted to replace mCherry, generating pCSE2W. For glutathione S-transferase (GST) pull-down experiments, GST-tagged SAMHD1 was expressed from pCAGGS . Haemagglutinin (HA)-tagged wild type or K11A mutant SAMHD1, Turbo-RFP (tRFP) or MKRPR-tRFP, as well as human karyopherins, were expressed from pLNCX2 based MLV vectors . Yellow fluorescence protein (YFP)-tagged wild type or mutant SAMHD1 proteins were expressed from the puromycin selectable MIGR-I based MLV vector pCMS28YFP . HIV-1 or MoMLV based GagPol expression plasmids and VSV-G encoding plasmid pMD.G have been described . Short hairpin RNAs targeting human karyopherins (Additional file 1: Table S1) were designed using the Clontech shRNA software (http://www.clontech.com/GB/Support) inserted into pSIREN.Retro-Q (Clontech) and expressed from the human U6 promoter as described before . Flag-tagged Vpx proteins from HIV-2ROD or SIVs from red-capped mangabey (RCM) or rhesus macaque (MAC) were expressed from HIV-1 based lentiviral vectors as described . Hexahistidine (6HIS)-tagged wild type or K6R, K11R, K27R, K29R, K33R, K48R and K63R ubiquitin was expressed from lentiviral vector pHRSIN-6HIS-Ubi-GFP .
Viral vector production
293 T cells were transfected in 10 cm plates at a confluence of ~80% with 3 μg viral vector plasmid, 2 μg GagPol encoding plasmid (p8.91 for HIV-1 based vectors, and pCMVi  for MLV based vectors) and 2 μg pMD.G using 4 μg polyethylenimine (PEI) per μg DNA in 1 ml of OptiMEM (Gibco) per 10 cm plate. The medium was changed 24 h post transfection, the virus was harvested at 48 h and 72 h post transfection, passed through a 0.45 μm filter and the collections were pooled. Depending on the experiment, viral vector supernatants were subjected to purification through a sucrose cushion .
Antibodies used for western blots were: mouse monoclonal SAMHD1 antibody 1 F9 at dilution 1/3000 (Abcam, ab117908), mouse monoclonal α-tubulin antibody DM1A at dilution 1/3000 (Sigma Aldrich, T9026), mouse monoclonal GST antibody at dilution 1/10000 , mouse monoclonal KPNB1 antibody 3E9 (Abcam, ab2811), rabbit polyclonal Hsp90 antibody H-114 at dilution 1/3000 (Santa Cruz Biotechnology, sc-7947), HRP-linked mouse monoclonal Flag antibody M2 at dilution 1/3000 (Sigma Aldrich, A8592) and HRP-linked rat monoclonal HA antibody 3 F10 at dilution 1/5000 (Roche Applied Science, 12013819001). For immunofluorescence staining mouse monoclonal HA antibody 12CA5 was used at dilution 1/250. For immunoprecipitation we used HA antibody 12CA5 at a dilution of and GFP antibody (Roche Applied Science, 11814460001) at a dilution of.
Immunofluorescence & microscopy
HeLa cells were stably transduced with LNCX2 based MLV vectors expressing HA-tagged wild type or K11A mutant SAMHD1 and selected with 100 μg/ml G418 for two weeks. Stable drug selected cells were seeded at ~50% density on coverslips in 24-well plates and fixed with 4% paraformaldehyde (PFA) 24 h after plating. Cells were permeabilised with 0.1% Triton X-100 washed twice in PBS and blocked in NGB buffer (50 mM NH4Cl, 1% goat serum, 1% bovine serum albumin) for 1 h, as described before . The cells were incubated with primary antibody diluted 1/250 in NGB buffer at room temperature for 1 h, washed three times in PBS and incubated for 1 h with species-specific Alexa Fluor 488- or 594-conjugated secondary antibody (Molecular Probes) diluted 1/500 in NGB buffer. Cells were washed three times in PBS and incubated with 4′,6′-diamidino-2-phenylindole (DAPI) dilactate (0.1 μg/ml, Molecular Probes) or Hoechst 33258 (1 μg/ml, Sigma Aldrich) for nuclei staining, washed again and mounted on glass slides using Mowiol (Calbiochem). For autofluorescence imaging, 293 T cells were stably transduced with pCMS28YFPWT or pCMS28YFPK11A encoding N-terminal YFP-tagged wild type or K11A mutant SAMHD1, respectively, and selected with 1 μg/ml puromycin for one week. Stably transduced cells were seeded on coverslips in 24-well plates, fixed using 4% paraformaldehyde and incubated with DAPI or Hoechst before mounting. For karyopherin knock down experiments, 293 T cells expressing wild type YFP-tagged SAMHD1 were seeded on coverslips as before and transduced once with MLV SIREN-RetroQ vector expressing shRNAs targeting the different karyopherins for 48 h before the cells were fixed and mounted as before. Laser scanning confocal microscopy was performed on a DM IRE2 microscope (Leica), and images were processed and analysed using LCS (Leica), ImageJ (NIH) and Photoshop (Adobe) software packages.
HeLa cells or MDM transduced with HA empty vector (EV) control were stained in parallel to SAMHD1WT and SAMHD1K11A transduced cells with anti-HA antibody as described above. From HA-EV control cells average MFI background stainings for the nuclear and cytoplasmic areas were calculated per pixel using ImageJ by defining an area of interest around the nucleus as defined by DAPI staining and around the whole cell (cytoplasmic MFI was derived by subtracting nuclear MFI from whole cell MFI). For HA-SAMHD1WT as well as HA-SAMHD1K11A expressing cells the pixel number of nuclear and whole cell areas was multiplied with the average background MFI per pixel for each area (as derived from the HA-EV control) and this value was subtracted from the integrated density (MFI × area) of the nuclear or cytoplasmic area for HA-SAMHD1 expressing cells using ImageJ. Percentage of nuclear SAMHD1 was calculated as the ratio of the integrated densities of nuclear SAMHD1 to whole cell associated SAMHD1.
293 T cells were seeded at 6 105 per well in 6-well plates and transfected the next day using PEI with 2 μg of each GST vector and HA–SAMHD1, HA-KPNA or Flag-Vpx vectors. The media was replaced 24 h post transfection. Cells were washed once in 1 ml PBS and then harvested in 1 ml lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 5% glycerol; 1% Triton X-100; Complete Protease inhibitor (Roche Diagnostics)) 48 h post transfection. Lysed cells were incubated on a rotating wheel at 4°C for 20 min, then clarified using a bench top centrifuge at 13,000 × g at 4°C for 15 min. For input analysis 50 μl were removed, mixed with 50 μl 2× Laemmli buffer and boiled. The remaining cell lysate was incubated with 25 μl glutathione-sepharose beads (50% solution in wash buffer) for 3 h at 4°C on a rotating wheel. Beads were washed three times in wash buffer (50 mM Tris-Hcl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 5% glycerol; 0.1% Triton X-100; Complete Protease Inhibitor (Roche Diagnostics)) and proteins were eluted by boiling in 50 μl 2× Laemmli buffer. Protein samples were analysed by SDS-PAGE and western blotting.
SAMHD1 degradation assay
293 T cells were seeded at 1 × 105 per well in 24-well plates and co-transfected with 100 ng pCMS28YFPSAMHD1WT or pCMS28YFPSAMHD1K11A plasmids and 100 to 1000 ng Flag-tagged VpxMAC, VpxRCM or VpxHIV-2ROD encoding plasmid. The percentage of YFP expressing cells, as well as the MFI were measured 24 h post transfection by flow cytometry using a FACS Calibur machine and CELLQuest software (BD). For SAMHD1 degradation assays in THP-1 we plated 2 105 × YFP-SAMHD1WT or YFP-SAMHD1K11A expressing cells into 48-well plate dishes and added sucrose-purified and concentrated VpxMAC containing or Vpx-deficient control VLPs corresponding to 10 ng reverse transcriptase (RT) or left cells untreated. After indicated time points cells were harvested and cell lysates were subjected to western blot analysis.
S35pulse chase labeling to measure SAMHD1 stability
293 T cells were seeded at 5 × 105 per well in 6-well plates and transfected the next day with 1 μg of YFP-SAMHD1WT, HA-SAMHD1WT or HA-SAMHD1K11A expressing plasmids using 4 μg polyethylenimine (PEI) per μg DNA in 0.5 ml of OptiMEM (Gibco) per well. Twenty-four hours later cells were washed twice in cysteine/methionine depleted DMEM (Gibco) and incubated for 20 min at 37°C in depletion media. The media was aspirated and replaced by pulse labeling media (depletion media containing 0.25 mCi/ ml S35 labeled cysteine/methionine; S35-Cys/Met EXPRESS, Perkin Elmer) for 10 min before trypsinising cells. Cells were washed three times in 10 ml DMEM and replated in DMEM into 6-well plate dishes. The 0 h time point was collected at the time of cell plating and subsequent time points were collected as indicated. Cells were lysed in 500 μl lysis buffer (50 mM Tris-HCL (pH7.6), 150 mM NaCl, 1% Triton X-100) and incubated on ice for 10 minutes. Cell lysates were pelleted at 1000 g for 10 minutes to remove insoluble material. Lysate supernatants were incubated with antibody (anti HA or anti GFP) -conjugated magnetic protein G beads (GE Healthcare) for 2 h at 4°C while rocking. Beads were washed three times in lysis buffer, eluted with Laemmli protein loading buffer, boiled and separated by 10% SDS PAGE. Gels were dried and exposed on a phosphor image screen over several days before development on a Typhoon Trio phosphorimager (GE Healthcare).
Ectopic gene expression in human monocyte derived macrophages
MDM were isolated and differentiated as described above , seeded in 48-well plates at 2 × 105 or 24-well plates for immunofluorescence at 5 × 106 cells per well, respectively. For ectopic gene expression, macrophages were treated with purified SIVMAC Vpx VLPs corresponding to 10 to 20 ng reverse transcriptase (RT) as measured by the colorimetric reverse transcriptase assay (Roche) and CSE2W, CSHAwtW or CSHAk11aW derived HIV-1 vector supernatants. Three days after transduction, the supernatants were harvested and tested for the presence of type I IFN using HEK-Blue™ cells and the transduced macrophages were challenged with at least four different doses of HIV-1 GFP reporter vector CSGW derived viral supernatants. At the time of infection with the GFP reporter virus parallel samples were harvested for western blot or fixed for immunofluorescence analysis. The infectivity of the GFP reporter virus was determined two days after infection by flow cytometry. Transduction efficiency of the MDM was evaluated by measuring percent of E2-Crimson positive cells using flow cytometry 72 h after transduction.
Pull down of hexahistidine-tagged proteins
Pull down of hexahistidine-ubiquitinated proteins in mammalian cells has been described . Briefly, 293 T cells were seeded at 6 × 105 cells per well in 6-well plates and transfected using PEI with 1 μg pHRSINGFP-6HIS -UbiWT or -UbiK-R mutant plasmids, MLV based vector encoding HA-tagged wild type or K11A SAMHD1 or empty vector control, as well as Flag-tagged VpxMAC or VpxRCM. The medium was replaced 24 h post transfection. Cells were washed once 48 h post transfection in 1 ml PBS and then harvested in 1 ml ice cold PBS. An aliquot was reserved as the input sample, mixed with 2× Laemmli buffer and boiled. The remaining cells were resuspended in 1 ml lysis buffer (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4, 10 mM imidazole, pH 8.0) and subjected to 10 s sonication using a small tip at level 2 before adding 50 μl of equilibrated (50%) NiNTA-agarose (Qiagen). The lysates were incubated rotating at room temperature for 3 h. The NiNTA-agarose was washed two times in 1 ml lysis buffer, two times in buffer L/TI (1 volume lysis buffer + 3 volumes buffer TI [25 mM Tris/HCl, 20 mM imidazole, pH 6.8]) and one time in buffer TI. Bound proteins were eluted by boiling in 100 μl 2× Laemmli buffer with 300 mM imidazole and separated by SDS-PAGE.
SAMHD1 sequence accession numbers
SAMHD1 sequences were derived from GenBank or Ensembl databases and had following accession numbers: Homo sapiens [GenBank: NM_015474.3], Pan troglodytes [GenBank: NM_001280510.1], Pongo abelii [GenBank: XM_002830274.2], Gorilla gorilla [GenBank: NM_001279619.1], Hylobates agilis [GenBank: JQ231127.1], Macaca mulatta [GenBank: NM_001271642.1], Nomascus leucogenys [GenBank: JQ231129.1], Callicebus molloch [GenBank: JQ231152.1], Nasalis larvatus [GenBank: JQ231144.1], Lagothrix lagotricha [GenBank: JQ231150.1], Callithrix jacchus [GenBank: JN936906.1], Alenopithecus nigroviridis [GenBank: JQ231142.1], Erythrocebus patas [GenBank: JQ231138.1], Chlorocebus tantalus [GenBank: JQ231136.1], Chlorocebus pygerythrus [GenBank: JQ231137.1], Aotus trivirgatus [GenBank: JQ231148.1], Cercopithecus neglectus [GenBank: JQ231141.1], Pteropus vampyrus [Ensembl: ENSPVAT00000010770], Myotis lucifugus [Ensembl: ENSMLUT00000002224], Echinops telfairi [Ensembl: ENSETET00000002844], Monodelphis domestica [GenBank: XM_001381548.2], Erinaceus europaeus [Ensembl: ENSEEUT00000004266], Anolis carolinensis [GenBank: XM_003220542.1], Gallus gallus [GenBank: NM_001030845.1], Rattus norvegicus [GenBank: NM_001191743.1], Mus musculus [GenBank: NM_018851.3], Oryctolagus cuniculus [Ensembl: ENSOCUT00000007611], Danio rerio [GenBank: NM_001159933.1], Felis catus [Ensembl: ENSFCAT00000012967], Canis familiaris [Ensembl: ENSCAFT00000013745], Ailuropoda melanoleuca [Ensembl: ENSAMET00000013697], Tursiops truncates [Ensembl: ENSTTRT00000001930], Ochotona princeps [Ensembl: ENSOPRT00000012314], Dipodomys ordii [Ensembl: ENSDORT00000012349], Otolemur garnettii [Ensembl: ENSOGAT00000000909], Microcebus murinus [Ensembl: ENSMICT00000002449], Equus caballus [Ensembl: ENSECAT00000009592], Bos taurus [Ensembl: ENSBTAT00000043682], Sorex araneus [Ensembl: ENSSART00000011255], Macropus eugenii [Ensembl: ENSMEUT00000000939].
We wish to thank Adam Fletcher, Paul Lehner, Juan Martin-Serrano, Chad Swanson, Greg Towers and Damien Warner for the generous provision of reagents and helpful discussions. This work was supported by the U.K. Medical Research Council, the European Commission’s Seventh Framework Programme [FP7/2007-2013] under grant agreements n°PIEF-GA-2009-237501 (to CG) and grant agreements n°PIEF-GA-2012-329679 (to DP), and the Department of Health via a National Institute for Health Research comprehensive Biomedical Research Centre award to Guy’s and St. Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust.
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