Methods

CRISPR/Cas9 knock-in strategy to evaluate phospho-regulation of SAMHD1

Cell lines

Human 293T/17 (ATCC No.: CRL-11268) cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) and 2 mM L-glutamine (Sigma-Aldrich) at 37°C and 5% CO2. Human BlaER1 cells (a kind gift of Thomas Graf) (29) cells were grown in RPMI (Sigma-Aldrich) supplemented with 10% FCS and 2 mM L-glutamine at 37°C and 5% CO2. For transdifferentiation, 1 × 106 BlaER1 cells per well of a 6-well tissue culture plate were treated with 10 ng/ml human recombinant M-CSF and IL-3 (PeproTech) and 100 nM β-estradiol (Sigma-Aldrich) for 7 days. Half of the cell culture supernatant was replaced with medium containing cytokines and β-estradiol at days 2 and 6. All cell lines were free of mycoplasma contamination, as tested by PCR Mycoplasma Test Kit II (PanReac AppliChem).

CRISPR/Cas9 knock-out and knock-in

For CRISPR/Cas9 mediated SAMHD1 knock-out (KO), 200 pmol Edit-R Modified Synthetic crRNA targeting SAMHD1 exon 1 (crSAMHD1ex1, target sequence: 5’-ATC GCA ACG GGG ACG CTT GG, Dharmacon), 200 pmol Edit-R CRISPR-Cas9 Synthetic tracrRNA (Dharmacon) and 40 pmol Cas9-NLS (QB3 Macrolab) were assembled _in-vitro, as previously described (51). Ribonucleoproteins were introduced into 1 × 106 sub-confluent BlaER1 cells using 4D-Nucleofector X Unit and SF Cell line Kit (Lonza), applying program DN-100. Single cell clones were generated using limited dilution one day after nucleofection. To confirm bi-allelic SAMHD1 KO, the modified region was amplified using primer SAMSeq_Gen-23_FW (5’-GAT TTG AGG ACG ACT GGA CTG C) and SAM_Seq_Gen1116_RV (5’-GTC AAC TGA ACA ACC CCA AGG T) together with GoTaq polymerase (Promega), followed by cloning into pGEM T-easy vector system (Promega) and Sanger sequencing. For knock-in (KI), 100 pmol of the respective ssDNA homologous recombination template with 30 bp homology arms to introduce T592A (5’-TAG GAT GGC GAT GTT ATA GCC CCA CTC ATA GCA CCT CAA AAA AAG GAA TGG AAC GAC AGT A, Dharmacon) or T592E (5’-TAG GAT GGC GAT GTT ATA GCC CCA CTC ATA GAA CCT CAA AAA AAG GAA TGG AAC GAC AGT AC) was nucleofected together with ribonucleoprotein complex containing crSAMHD1_ex16 (target sequence: 5’-TTT TTT TGA GGT GTT ATG AG, Dharmacon). When single cell clones reached confluency, duplicates were generated. One half was lysed (10 min, 65°C; 15 min, 95°C) in lysis buffer (0.2 mg/ml Proteinase K, 1 mM CaCl2, 3 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 10 mM Tris (pH 7.5)) (52) and screened for successful KI using mutation specific custom designed KASP genotyping assays (LGC) and KASP V4.0 2x Master mix (LGC) on a CFX384 Touch Real-Time PCR Detection System (BioRad). Homozygous KI was confirmed by Sanger sequencing after amplification using primer SAM_Seq_Gen58570_FW (5’-CAT GAA GGC TCT TCC TGC GTA A) and SAM_Seq_Gen59708_RV (5’-ACA AGA GGC GGC TTT ATG TTC C) together with KOD Hot Start DNA Polymerase (Merck). Additionally, allele specific sequencing as described for SAMHD1 KO was performed, if required. Presence of large deletions in the region between amplification primers was excluded by PCR and analytic gel electrophoresis. Presence of both alleles was confirmed by quantitative genomic PCR (30), performed using _SAMHD1 exon 16 specific PrimeTime qPCR Assay (FW: 5’-CTG GAT TGA GGA CAG CTA GAA G, RV: 5’-CAG CAT GCG TGT ACA TTC AAA, Probe: /56-FAM/ AAA TCC AAC /Zen/ TCG CCT CCG AGA AGC /3IABkFQ/, IDT), human TERT TaqMan Copy Number Reference (Thermo Fischer) and PrimeTime Gene Expression Master Mix (IDT) on a CFX384 machine.

HIV-1 reporter virus infection

VSV-G pseudotyped HIV-1 reporter viruses pNL4.3 E− R− luc (53) (HIV-1-luc) and pNL4.3 IRES mCherry E− R+ (HIV-1-mCherry) were produced, as detailed previously (22). Briefly, pNL4.3 E− R− luc (a kind gift of Nathaniel Landau) or pNL4.3 IRES mCherry E− R+ (a kind gift of Frank Kirchhoff) were co-transfected together with pCMV-VSV-G into 293T/17 cells using 18 mM polyethylenimine (Sigma-Aldrich). Filtered (0.45 μm) supernatants were treated with 1 U/ml DNAse I (NEB; 1 h, RT) and purified through a 20% sucrose cushion (2 h, 106750g, 4°C). Viral stocks were titrated for β-galactosidase activity on TZM-bl cells. Virus-like particles containing Vpx (VLP-Vpx) were produced in an analogue manner using pSIV3+ (54) derived from SIVmac251 (a kind gift of Nicolas Manel) and pCMV-VSV-G. The amount of VLP-Vpx used in all experiments was optimized for complete SAMHD1 degradation. For infection 3 × 104 cells were seeded per well of a 96-well tissue culture plate. Transdifferentiated BlaER1 cells were allowed to settle for 2 h in medium without cytokines and β-estradiol. VSV-G pseudotyped HIV-1 reporter virus at indicated MOI, as well as VLP-Vpx, were added, followed by spin occulation (1.5 h, 200g, 32°C). Infection was quantified after 24 h by FACS (for HIV-1-mCherry) or by adding 50 μl/well britelite plus reagent (PerkinElmer) and measurement on a Pherastar FS (BMG) (for HIV-1-luc). To show VLP-Vpx mediated SAMHD1 degradation, 4.4 × 105 transdifferentiated BlaER1 cells were treated in a 12-well tissue culture plate in the same conditions and concentrations as stated above.

Flow Cytometry

For FACS analysis of BlaER1 transdifferentiation, 1 × 106 native or transdifferentiated BlaER1 cells were collected, washed once in FACS buffer (10% FCS, 0.1% Sodium acetate in PBS; 10 min, 300g, 4°C) and stained with CD11b-APC (M1/70, Biolegend), CD19-PE (HIB19, Biolegend) or respective isotype controls (Biolegend) and Fixable Viability Dye eFluor 780 (Thermo Fischer) in presence of FC Block (BD, 20 min, 4°C). Stained cells were washed in FACS buffer twice and fixed in 2% paraformaldehyde (30 min, RT), before analyzing on a LSR II instrument (BD). For readout of HIV-1-mCherry infection, six wells of a 96-well plate were pooled and stained with CD11b-APC and Fixable Viability Dye eFluor 780 as detailed above. Infected cells were analyzed on a BD LSRFortessa.

Immunoblot

For immunoblot, cells were washed in PBS, lysed in radioimmunoprecipitation buffer (RIPA; 2 mM EDTA, 1% glycerol, 137 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, 25 mM Tris (pH 8.0)) supplemented with proteinase and phosphatase inhibitor (Roche) for 30 min on ice. Lysate was cleared (30 min, 15000g, 4°C) and protein content was measured by Bradford assay using Protein Assay Dye Reagent Concentrate (BioRad). 20 μg total protein were denatured (10 min, 70°C) in NuPAGE LDS Sample Buffer and Reducing Reagent (Thermo Fischer) and separated on a NuPAGE 4-12% Bis-Tris gradient gel (Thermo Fischer) in MOPS running buffer (1 M MOPS, 1 M Tris, 69.3 mM SDS, 20.5 mM EDTA Titriplex II). Transfer was performed in an XCell II Blot Module in NuPAGE Transfer Buffer (Thermo Fischer) onto a Hybond P 0.45 PVDF membrane (GE Healthcare). After blocking in 5% BSA or milk powder (Carl Roth) in TBST (Tris-buffered saline, 0.1% Tween; 2 h, 4°C), primary antibodies anti-GAPDH (14C10, CST), anti-Cyclin B1 (4138, CST), anti-Cyclin A2 (4656, CST), anti-SAMHD1 (12586-1-AP, Proteintech), anti-SAMHD1 (A303-691A, Bethyl) and anti-SAMHD1-pT592 (D702M, CST) diluted in 5% BSA or milk powder in TBST were applied overnight at 4°C. Subsequent to washing in TBST, anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody (CST) was applied (2 h, 4°C) and the membrane was washed again before detection on a FUSION FX7 (Vilber Lourmat) using ECL Prime reagent (GE). If required, membranes were stripped of bound antibody in stripping buffer (2% SDS, 62.5 mM Tris-HCl (pH 6.8), 100 mM β-mercaptoethanol; 1 h, 65°C). Band densities were determined with FUSION software (Vilber Lourmat).

Cellular dNTP levels and concentrations

For measurement of cellular dNTP levels, 2 × 106 transdifferentiated BlaER1 cells were washed in PBS and subjected to methanol extraction of dNTPs, followed by quantification of all four dNTPs by single nucleotide incorporation assay, as described previously (31). CD19 depletion was performed using CD19 microbeads and MS columns (Miltenyi). Cell volumes were determined by seeding respective cell types on a Poly-D-Lysine (Sigma) coated (10%, 1.5h, RT) Cell Carrier-96 well plate (Perkin Elmer). After centrifugation (5 min, 300g), cells were fixed (4% PFA, 15 min, 37°C), permeabilized (0.1% Triton X-100, 5 min, 37°C) and stained using HCS CellMask Deep Red Stain (Thermo Fischer, 30 min, RT). Z-Stack of stained cells was acquired using confocal imaging platform Operetta (Perkin Elmer) and volume was calculated as a sum of cell areas in all relevant Z-stacks using Harmony software (Perkin Elmer).

Statistical analysis

Statistical analysis was performed using GraphPad Prism (V8). Mean and standard deviations are shown. Statistical significance was assessed using unpaired two-tailed t-test, as well as non-parametric Kruskal-Wallis test or parametric One-Way ANOWA, corrected against multiple testing using Dunn’s or Dunnet correction, respectively.

Article TitleCRISPR/Cas9 knock-in strategy to evaluate phospho-regulation of SAMHD1

Abstract

Sterile α motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. As a dNTP triphosphate triphosphohydrolase (dNTPase), SAMHD1 is proposed to limit cellular dNTP levels correlating with inhibition of HIV-1 reverse transcription. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is also not completely understood.

We use BlaER1 cells as a novel human macrophage transdifferentiation model combined with CRISPR/Cas9 knock-in (KI) to study SAMHD1 mutations in a physiological context. Transdifferentiated BlaER1 cells, resembling primary human macrophages, harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. Co-delivery of Vpx or CRISPR/Cas9-mediated SAMHD1 knock-out relieves the block to HIV-1. Using CRISPR/Cas9-mediated homologous recombination, we introduced specific mutations into the genomic SAMHD1 locus. Homozygous T592E mutation, but not T592A, leads to loss of HIV-1 restriction, confirming the role of T592 dephosphorylation in the regulation of anti-viral activity. However, T592E KI cells retain wild type dNTP levels, suggesting the antiviral state might not only rely on dNTP depletion.

In conclusion, the role of the T592 phospho-site for anti-viral restriction was confirmed in an endogenous physiological context. Importantly, loss of restriction in T592E mutant cells does not correlate with increased dNTP levels, indicating that the regulation of anti-viral and dNTPase activity of SAMHD1 might be uncoupled.

Importance Sterile α motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent anti-viral restriction factor, active against a broad range of DNA viruses and retroviruses. In myeloid and resting CD4+ T cells, SAMHD1 blocks reverse transcription of immunodeficiency virus 1 (HIV-1), not only inhibiting viral replication in these cell types, but also limiting the availability of reverse transcription products for innate sensing of HIV-1. Manipulating SAMHD1 activity could be an attractive approach to improve HIV-1 therapy or vaccination strategies. Anti-viral activity is strictly dependent on dephosphorylation of SAMHD1 residue T592, however the mechanistic consequence of T592 phosphorylation is still unclear. Here, we use BlaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated KI to study the influence of SAMHD1 T592 phosphorylation on anti-viral restriction and control of cellular dNTP levels in an endogenous context. By using this novel approach, we were able to genetically uncouple SAMHD1’s anti-viral and dNTPase activity with regard to regulation by T592 phosphorylation. This suggests that SAMHD1 dNTPase activity may not exclusively be responsible for the anti-lentiviral activity of SAMHD1 in myeloid cells. In addition, our toolkit may inspire further genetic analysis and investigation of SAMHD1-mediated restriction, as wells as its cellular function and regulation, leading to a deeper understanding of SAMHD1 and HIV-1 biology.


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