Epigenetic silencing by the SMC5/6 complex mediates HIV-1 latency

Cell lines and primary culturesImmortalized cell lines

Human 293T cells (female) were initially purchased from the American Type Culture Collection (ATCC) and were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Hyclone) and an antibiotic-antimycotic (Gibco).

Human CEM-SS cells (female) were obtained from the NIH AIDS Reagent and were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS and antibiotic-antimycotic.

CEM-SS cells stably expressing Cas9 protein were produced using a modified pLentiCrispr v2-Blast plasmid that was a gift from Mohan Babu (Addgene plasmid 83480). The U6-promoter, sgRNA scaffold and EF1-α promoter were excised from pLentiCrispr v2-Blast by cleavage with KpnI and AgeI and replaced with an SFFV promoter. Lentiviruses were made from this construct by transfecting 5 × 106 293T cells in a 15 cm dish with 15 µg of the lentiviral vector, as well as 10 µg and 5 µg of the packaging plasmids pCMVR8.74 and pMD2.G, respectively, using polyethylenimine (PEI). The media were changed 24 h post transfection (hpt). Supernatants containing lentiviral particles were collected at 72 hpt, filtered through a 0.44 µm filter and run through a 100,000 MWCO concentrator (Amicon). Following concentration, 5 × 106 CEM-SS cells were incubated with 2 ml of the concentrated supernatant at 37 °C overnight. The media were then replaced with fresh RPMI medium and cells incubated for 48 h. At this point, the media were replaced with fresh RPMI medium supplemented with 20 µg ml−1 of blasticidin (Santa Cruz) to allow selection of transduced cells. Cells were then single-cell cloned by aliquoting limited dilution in 96-well plates such that each well has ~10% chance of having a cell in it. These cloned cells were then analysed for Cas9 activity.

Primary CD4+ T cells

Human blood from healthy donors was purchased from the Gulf Coast Regional Blood Center. All donors tested negative for HIV-1 and HIV-2. Samples were de-identified before purchasing. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density-gradient centrifugation over Histopaque (Sigma) and CD4+ cells isolated using a CD4+ isolation kit (Invitrogen). Isolated CD4+ cells were activated by incubation with antibodies against CD28/CD49d (BD Biosciences) and 5 µg ml−1 phytohaemagglutinin (PHA) in RPMI supplemented with 10% FBS and IL-2 as previously described47. Cells were maintained at 105–106 cells per ml in the presence of IL-2, CD28/CD49d antibodies and PHA for 1 week before infection with HIV-1.

HIV-1 production

An HIV-1 nano luciferase reporter virus (NL-NLuc) was generated from the parental NL4-3 virus by substituting the viral nef gene in NL4-3 with the NLuc indicator gene48. NL-NLucΔEnv was made from NL-NLuc by removal of a 943 bp segment of the env gene that makes it replication incompetent. Similarly, the GFP reporter virus (NL-GFPΔEnv) was generated from NL-NLucΔEnv by substituting GFP in place of nef. The NL-DC reporter virus expresses eGFP under the control of the HIV-1 LTR and mCherry from an internal EF1-α promoter and was generated by cloning an EF1-α:mCherry cassette into the XhoI site located 3′ to GFP in NL-GFPΔEnv. All these reporter viruses have an intact vpr gene.

A GFP reporter virus, IN− NL-GFPΔVpr, lacking a functional Vpr protein, was created from the parental IN− NL-GFP virus by inserting a TTAA duplication at 15 bp 3′ of the Vif stop codon. This introduces a stop codon and a frameshift mutation early in the Vpr open reading frame that creates a non-functional truncated Vpr protein49. These viruses either have WT integrase (IN+) or contain the D64V (IN−) mutation that blocks IN function.

Plasmids expressing the replication-competent NL-NLuc provirus were transfected into 293T cells using PEI. Non-spreading NL-NLucΔEnv and NL-GFPΔEnv proviruses were co-transfected into 293T cells with the pMD2.G plasmid encoding the VSV-G protein. After 24 h, the spent media were replaced with fresh media. At 72 hpt, supernatant media were filtered through a 0.44 μm filter. WT or IN− HIV-1-containing supernatant media were normalized by p24 levels, measured by ELISA, before being used to infect target cells. The MOI:p24-normalized volume ratio was assessed by infecting 106 CEM-SS cells with IN+ NL-GFPΔEnv, or a CEM-SS inducible Tax cell line (expressing an HTLV-1 Tax protein that is known to activate gene expression from unintegrated HIV-110) with IN− NL-GFPΔEnv. These cells were infected with varying dilutions of the virus, then analysed by flow cytometry at 2 dpi (gating strategy outlined in Extended Data Fig. 9). The number of GFP+ cells at each dilution was then converted to MOI using the formula MOI = −ln (1− proportion of GFP+ cells) which was then correlated to the p24 level in the viral stock. Thus, the amount of IN− virus used (per million cells) for each MOI could be determined.

CRISPR knockout screen

The Brunello human CRISPR knockout library (Addgene 73178)15 was used to transform electrocompetent cells (Endura). Of the library (resuspended in water), 500 ng was used to transform a total of 4 × 25 μl of cells in a 1 mm cuvette (10 μF, 600 Ω, 1.8 kV) to yield >108 colonies when plated to ensure each sgRNA in the library was covered ~1,000× on average. These colonies were collected and the pooled library plasmids extracted using Maxiprep columns (Zymo).

Lentiviral libraries were created by transfecting 293T cells with library DNA and the packaging plasmids pCMVR8.74 and pMD2.G using PEI. The media were changed at 24 hpt, and the supernatant containing the lentivirus library was collected and filtered through a 0.44 µm filter at 72 hpt. The lentivirus was titrated and used to transduce CEM-SS Cas9 cells that were then subjected to puromycin (Gemini) kill curves at 2 dpt to determine the amount of lentivirus that correlated to an MOI of 0.3. This amount was then used to transduce 108 CEM-SS Cas9 cells at 0.3 MOI, and the cells were selected in 1 µg ml−1 puromycin at 2 dpt for a week.

The pooled knockout cells were then infected for 2 d with an IN− HIV-1 NL-GFP reporter virus that has an inactivating D64V amino acid substitution in the integrase gene. These infected cells were then run through a BSL-3 contained FACS Aria II (BD Biosciences) to collect the GFP+ cell population from which genomic DNA was extracted. Purified gDNA was incubated with the restriction enzyme DpnI to remove any residual plasmid contamination.

The sgRNA from this DNA was amplified by PCR using flanking primers (FP: 5′-TGGACTATCATATGCTTACCGTAACTTGA -3′ RP: 5′-GGCTCGAGGGGGCCCGGGTGCAAAGATGGATA -3′) and then cloned back into the parental pLentiGuide Puro plasmid via the NdeI and XmaI restriction sites. Subsequent rounds of transformation, lentivirus production, transduction and infection were carried out as described for a total of 3 rounds. In the final round, the DNA was amplified using the indexed Illumina sequencing primers, purified in a PCR purification spin column (Zymo) and sequenced on the NovaSeq 6000.

Sequencing data were analysed using MAGECK-VISPR50 by first generating sgRNA read counts via invoking ‘mageck count’, analysing sgRNA enrichment and getting gene ranks using the Robust Rank Aggregation algorithm on normalized read counts. ‘Mageck test’ was run with ‘–remove-zero both–remove-zero-threshold 0’ parameters as previously suggested51.

CRISPR single-gene knockouts

Single-gene knockout cells were generated by transducing CEM-SS Cas9 cells with lentiviruses made from a pLentiGuide-Puro plasmid (Addgene 52963)52 expressing the sgRNA of interest. Transduced cells were selected at 2 dpt with 1 µg ml−1 puromycin for 1 week. Infection experiments on polyclonal knockout cells were carried out by infecting cells at this stage.

Clonal cells were isolated by aliquoting puromycin-resistant cells at limiting dilution into a 96-well plate, subsequent isolation and expansion. Knockout cells were then identified and validated by clonal sequencing of the genetic lesions and by western blot.

Western blot analyses

Cells were collected and lysed in Laemmli buffer, sonicated and denatured at 95 °C for 15 min. Lysates were subjected to electrophoresis on 4–20% SDS–polyacrylamide gels (Bio-Rad), transferred onto nitrocellulose membranes and then blocked in 5% milk in PBS + 0.1% Tween. Membranes were incubated in primary and secondary antibodies diluted in 5% milk in PBS + 0.1% Tween for 2 h each and then washed in PBS + 0.1% Tween. The membranes were incubated with a luminol-based enhanced chemiluminescent substrate and signals were visualized using GeneSnap (Syngene). The membranes were immunoblotted with specific antibodies to probe for SMC5 (Research Resource Identifier RRID:AB_2900565), SMC6 (RRID:AB_2747157), NSMCE2 (RRID:AB_10637854), NSMCE4A (RRID:AB_11169701), SLF1 (RRID:AB_10816722), SLF2 (RRID:AB_11129755), FLAG (RRID:AB_259529) or actin (RRID:AB_2687938). Primary antibodies were used at 1:1,000 dilution, except for the actin antibody which was used at 1:5,000. Secondary HRP-conjugated anti-mouse (RRID:AB258431) or anti-rabbit (RRID:AB_258284) antibodies were used at 1:5,000 dilution.

Flow cytometry

Cells were collected, washed in PBS and fixed in 1% paraformaldehyde in PBS for 10 min before being resuspended in 2% bovine serum albumin (BSA) in PBS and run through a cell strainer. Cells were run through a Fortessa X20 flow cytometer (BD Biosciences) and the data analysed using FlowJo v10.6.2.

Luciferase assay

Cells were collected, washed three times in PBS, lysed in passive lysis buffer (Promega) and assayed for NLuc activity using the Nano-Glo luciferase assay on a Lumat LB9507 luminometer (Bertold Technologies).

Quantification of HIV-1 replication and spread

Cells (107) from the parental Cas9 cell line, or the ΔSMC5 and ΔSLF2 cell lines, were infected with either WT or IN− NL-NLuc virus in a total of 20 ml RPMI. Viral stocks were pretreated with 5 U ml−1 DNase I to remove residual plasmid DNA, and all IN− infections were carried out in the presence of 20 µM raltegravir to prevent revertant mutations. Cells were counted at days 1, 2, 3, 5, 7, 10, 12, 14 and 16 dpi where possible, and media were periodically refreshed to maintain cell counts below 106 cells per ml. Live cells (106) were collected at each time point (where possible) and equally split to assay NLuc and for DNA and RNA extraction.

For DNA analysis, cells were pelleted and washed three times in ice-cold PBS. DNA was then extracted using DNA Miniprep Plus columns (Zymo) according to the manufacturer’s instructions, then incubated with DpnI (NEB) to remove any residual plasmid contamination.

For RNA analysis, cells were lysed in TRIzol (Thermo Fisher) to collect the RNA, and DNAse I treated to remove residual DNA contamination. The RNA was then converted to complementary DNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

Quantification of total HIV-1 DNA and RNA was carried out on a QuantStudio 6 Pro real-time qPCR machine (Thermo Fisher) using a custom total HIV-1 TaqMan probe that amplifies the U5-gag region on HIV-1.

For Alu-LTR real time nested qPCR, DNA was amplified using a nested PCR approach18. Briefly, an initial non-saturating PCR using primers ALU1 (5′-TCCCAGCTACTGGGGAGGCTGAGG-3′), ALU2 (5′-GCCTCCCAAAGTGCTGGGATTACA-3′) and L-HIV (5′- ATGCCACGTAAGCGAAACTTAAGCCTCAATAAAGCTTGC-3′) was performed using DNA isolated from HIV-1 infected cells. After the PCR products were purified using a PCR Kleen kit (Bio-Rad), nested qPCR was performed using primers AA55M (5′- GCTAGAGATTTTCCACACTGACTAA-3′) and L (5′- ATGCCACGTAAGCGAAAC-3′) and the SYBR green master mix (Thermo Fisher).

The amounts of unintegrated 2LTR HIV-1 circular DNA were quantified by qPCR using TaqMan primers/probes that amplify across the U5-U3 junction only present in 2LTR circles (FP: 5′- AACTAGGGAACCCACTGCTTAAG -3′, RP: 5′- TCCACAGATCAAGGATATCTTGTC -3′, probe: 5′- FAM- ACACTACTTGAAGCACTCAAGGCAAGCTTT -TAMRA-3′)53.

Relative quantification using the ΔΔCT method with β-Actin as an internal control was then carried out using either a genomic β-Actin (DNA) or spliced β-Actin (RNA) probe. Relative quantification using the ΔΔCT method54 with β-Actin as an internal control was then carried out.

ChIP–qPCR

The indicated cells were cultured at 106 cells per ml in RPMI and infected with IN− NL-GFP. Viral stocks were pre-incubated with 5 U ml−1 DNase I to remove plasmid contamination before infection.

Cells were collected at the indicated times post infection, rinsed twice with PBS and crosslinked with 1% formaldehyde for 15 min at 25 °C before being quenched in 0.125 M glycine for 5 min. The rinsed cells were then lysed in ChIP lysis buffer (50 mM Tris-HCl pH 8.0, 1% sodium dodecyl sulfate, 10 mM EDTA) and sonicated on ice with a Fisher Sonic Dismembrator 60 (output 4.5, 20 s pulse repeated 6 times on ice with 40 s between each sonication). The supernatant containing sonicated chromatin was pre-cleared by the addition of magnetic Protein G dynabeads (Thermo Fisher) that had been pretreated with denatured salmon sperm DNA (Invitrogen). The magnetic beads were removed, and the sonicated chromatin was incubated overnight at 4 °C using 2.5 µg of the indicated antibody in ChIP dilution buffer (16.7 mM Tris-HCL pH 8.0, 1% Triton X-100, 0.01% SDS, 150 mM NaCl, 1.2 mM EDTA). The sonicated chromatin (5%) was stored as input DNA without further treatment until the reverse crosslinking step.

Protein G dynabeads were then added to the chromatin-antibody mixture, incubated for 2 h at 4 °C and then washed 3 times with ChIP LiCl buffer (10 mM Tris-HCL pH 8.0, 1% NP-40, 250 mM LiCl, 1 mM EDTA, 1% Na deoxycholate) and twice with TE buffer (10 mM Tris-HCL pH 8.0, 1 mM EDTA). Protein-DNA complexes were eluted from the beads with an elution buffer (0.1 M NaHCO3, 1% SDS), de-crosslinked by incubating at 65 °C for 16 h and at 95 °C for 15 min, then digested by adding 50 μg proteinase K and incubating at 50 °C for 3 h. DNA was extracted using a DNA Miniprep Plus kit (Zymo), digested with DpnI (NEB) to remove any plasmid contamination, then used for qPCR analysis using primers that amplify U5-R on the HIV-1 promoter (FP: 5′- CTCTCTGGTTAGACCAGATC-3′, RP: 5′-GCTAGAGATTTTCCACACTG-3′). ChIP data are expressed as a percentage of input DNA.

Rescue of ΔNSMCE2 knockout cells

A lentiviral vector expressing a FLAG-tagged NSMCE2 protein that is resistant to cutting by the sgRNA expressed in the CEM-SS ΔNSMCE2 knockout cells was created by mutating the NSMCE2 sequence from an expression plasmid (OHu31586, GenScript) via overlap extension PCR to introduce synonymous T-C and C-T mutations into the sgRNA target sequence (GTATCAACTCTGGTATGGAC to GcATtAACTCTGGTATGGAC). This mutant FLAG-NSMCE2 PCR product was then cloned into the pLCE lentiviral vector using NheI and XhoI restriction sites.

Similarly, the NSMCE2ΔSUMO mutant was created using overlap extension PCR to introduce the C185S and H187Q amino acid substitutions into the RING domain necessary for E3 SUMO ligase function25.

Lentiviruses were created from pLCE (control), pLCE FLAG-NSMCE2 and pLCE FLAG-NSMCE2ΔSUMO, which were then used to transduce CEM-SS or CEM-SS ΔNSMCE2 cells. These cells were infected with IN+/IN− NL-NLuc or IN− NL-GFP at 0.3 MOI and the cells were collected at 2 dpi for the respective NLuc assays (NL-NLuc), or for flow cytometry and ChIP–qPCR (NL-GFP). Expression of these NSMCE2 constructs was validated by western blot.

E3 SUMO ligase inhibition kinetics

TAK-981 (MedChemExpress) is a global inhibitor of SUMOylation26 and was reconstituted in DMSO to a 5 mM stock. TAK-981 was added to CEM-SS Cas9 and ΔSMC5 cells infected with NL-NLuc at 0, 5, 15, 30, 75, 150, 500 and 1,000 nM concentrations, and the cells were collected for an NLuc assay after 2 dpi.

To assay the effects of TAK-981 on viral RNA expression in infected cells, CEM-SS cells were infected with IN+/IN− NL-NLuc in the presence or absence of 150 nM TAK-981. RNA was extracted at 2 dpi with TRIzol (Thermo Fisher) and DNAse I treated for 2 h. cDNA was then made using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). The levels of unspliced HIV-1 RNA gag or the spliced viral RNA from donor 1 acceptor 1 (A1D1) and splice donor 7 acceptor 4 (A4D7) were quantified by qPCR using the primers for gag (FP: 5′-GCGAGAGCGTCGGTATTAAGCG-3′, RP: 5′-AATCGTTCTAGCTCCCTGCTTGC-3′), A1D1 (FP: 5′-GATCTCTCGACGCAGGACTC-3′, RP: 5′-TGGTCCTTTCCAAACTGGAT-3′) and A4D7 (FP: 5′- CAAGCTTCTCTATCAAAGCAACC -3′, RP: 5’- AATCGAATGGATCTGTCTCTGTC -3′)55.

To understand the kinetics of inhibiting SUMOylation on viral gene expression and epigenetic modifications in infected cells, 150 nM TAK-981 was added to CEM-SS Cas9 and ΔSMC5 cells at the time of infection, or at 16, 19, 21, 24, 36 and 48 hpi. Cells were infected with NL-NLuc Δenv and collected at 72 hpi for either NLuc assays or ChIP–qPCR using anti-SUMO2/3, H3Ac or H3K9me3 antibodies and quantifying the HIV-1 promoter using primers that amplify the U5-R region (FP: 5’- CTCTCTGGTTAGACCAGATC-3′, RP: 5’-GCTAGAGATTTTCCACACTG-3′). ChIP data are expressed as a percentage of input DNA.

HIV-1 latency quantificationCEM-SS latency assay

WT or ΔSMC5 CEM-SS cells were infected with the IN+ NL-GFPΔEnv virus at an MOI of ~0.1 in the presence or absence of 150 nM TAK-981. Nevirapine was added to the cells at 2 dpi to inhibit any late infection events, and at 3 dpi the cells were washed and resuspended in 2% BSA in PBS for sorting on a BSL-3 contained FACS Aria II (BD Biosciences) to isolate GFP− cells. These cells were allowed to recover in growth media for 6 d (9 dpi) before treatment with DMSO, 150 nM TAK-981, 80 nM PMA or 1 ng ml−1 TNF-α. Cells were collected the next day (10 dpi) to analyse GFP expression by flow cytometry.

CEM-SS delayed integration latency assay

WT or ΔSMC5 CEM-SS cells were infected with the IN+ NL-GFPΔEnv virus in the presence or absence of 500 nM raltegravir. At 2 dpi, all cells were washed three times with PBS, replated in RPMI with nevirapine and cultured for an additional 3 d. At 5 dpi, GFP− cells were sorted, allowed to recover in growth media for 6 d, treated with DMSO, PMA or TNF-α (11 dpi) and analysed for flow cytometry the next day (12 dpi).

Both CEM-SS control and ΔSMC5 cells were infected with a normalized p24 amount that was previously titred to give 10% GFP+ cells in CEM-SS cells +Ral (~0.45 MOI) and −Ral (0.1 MOI) at 5 dpi.

Primary activated CD4+ T cell latency assay

Activated primary T cells isolated from PBMCs were infected at a low MOI of ~0.05 with an IN+ NL-DC reporter virus that expresses eGFP off the HIV-1 LTR and mCherry from an internal EF1-α promoter. Infections were carried out in the presence and absence of 150 nM TAK-981 added at infection, or with 80 nM PMA known to reverse epigenetic silencing of HIV-1 in latent cells56 or 150 nM TAK-981 added 24 h before collection. Cells were collected for flow cytometry at 7 dpi in 2% BSA in PBS to measure eGFP and mCherry fluorescence. The CD4+ T cell population was separated from dead cells and cellular debris by gating on forward and side-scatter analyses. The high enrichment by the CD4 Positive Isolation kit (Invitrogen) also allowed us to identify the enriched CD4+ T cell population and gating was done to exclude the few possible CD4+ monocytes that might be present. Flow cytometry data were then analysed using FlowJo v10.6.2.

DNA was also extracted from these cells at 2, 3, 5 and 7 dpi, incubated with DpnI to remove residual HIV-1 plasmid contaminants, then the amounts of unintegrated 2LTR HIV-1 circular DNA quantified by qPCR as described in a previous section.

Statistical analysis

Sample size and P values are indicated in the text or figure legends. Error bars in the experiments represent standard deviations of the mean from independent experiments. Statistical analyses were performed using GraphPad Prism or reported by the indicated computational tools used in the analysis of the CRISPR knockout screen. Information about statistical methods is specified in the figure legends.

Materials availability

All unique materials generated in this study will be made available upon reasonable request to the lead contact and will also be made available through the NIH AIDS Reagent programme.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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