Vectors: The H1sgRNA (Control), dCas9-VPR and sgf2-362 have been previously described [7]. The amino acids sequence of ZFP-362 and ZFP-VEGF that bind their respective target sites were identified using the ZF Tools Ver 3.0 [47] and fused to VPR activation domain and cloned into pcDNA3.1 mammalian expression vector by Genscript. The ZFP-362 was ordered as a gBLOCK (IDT, IA, USA) and was clone into a dCas9-VPR vector (addgene #63798) to replace the dCas9 in this vector using NEBuilder® HiFi DNA assembly Master mix (NEB, MA, USA). The LTR-driven luciferase from different subtypes were obtained from the NIH AIDS Reagent Program (Cat. No. 4787, 4788, 4789, 4790, 4791, 4792, 4793, NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: pBlue3'LTR-luc-A from Dr. Reink Jeeninga and Dr. Ben Berkhout [48, 49].
Cell Lines: pMo-HEK and pΔ362 lentiviral vectors were generated as described in Shrivastava et al. [50]. HEK293T cells (ATCC) were transduced at a MOI of 0.1 to ensure a single integrated copy. 7 days post transduction, GFP positive cells were FACS sorted (FACSCalibur II, Becton, Dickinson and Company, East Rutherford, NJ at the Scripps Flow Cytometry core facility) to generate a cell line with a stably integrated HIV LTR or with a Δ362LTR that contains mutations at the 362 site. The J-Lat cell lines were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: J-Lat Full Length Clones 6.3, 10.6, and 15.4, which are collectivity referred to as J-Lat cells, which were deposited by Dr. Eric Verdin [51]. The LChIT reporter line, a CEM T-cell–based reporter system, comprising of the single-copy LTR integrant driving the expression of mCherry-IRES-tat has been described previously [52].
Cell Culture
HEK293T cells (ATCC) and the derived pMo-HEK and pΔ362 reporter lines were maintained in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, M, USA) and 50 µg/ml Pen/Strep (Thermo Fisher Scientific, Waltham, MA) at 37 °C and 5% CO2. J-Lat and LChIT cell lines were both maintained in RPMI 1640 (Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum, 2 mmol/l l-glutamine (Thermo Fisher Scientific, MA, USA), and 50 µg/ml Pen/Strep (Thermo Fisher Scientific, MA, USA) at 37 °C and 5% CO2.
CLADE-specific activation assays. HEK293 cells were transfected in triplicate using Lipofectamine 3000® (Thermo fisher scientific, MA, USA) with pcDNA-ZFP-362-VPR or pcDNA-VEGF-VPR with vectors expressing Firefly luciferase off the LTRs from different subtypes of HIV. A vector expressing Renilla luciferase was included as a background control (pRL-CMV, Promega, WI, USA). At 48 hrs post-transfection, a Dual-luciferase® Reporter Assay was performed according to manufacturer instructions and luciferase activity detected on the Glomax® Explorer system (Promega, WI, USA). The levels of Firefly luciferase were normalized to Renilla luciferase, and made relative to the pcDNA-VEGF-VPR control.
Activation of the HIV LTR: The protocol to assess the ability of the ZFP-362-VPR construct to activate latent HIV-1 has been described in Saayman et al. [52]. Briefly, pcDNA-ZFP-362-VPR, pcDNA-ZFP-VEGF-VPR (control), or pcDNA3.1 (Mock) plasmids were transfected into the CEM LChIT cells and the J-Lat cells using the Neon Transfection System (Thermo Fisher Scientific, MA, USA) at a cell density of 2 × 107 cells/ml with the following electroporation parameters: 3 pulses, 1,350 V and 10 ms. The dCas9-VPR + sgF2-362 vectors, which have been previously shown to potently activate HIV, were included as a comparison [52]. The cells were cultured without antibiotics following electroporation. To determine the activation with LRAs, J-Lat cells were treated with TNF-alpha (10 ng/ml, Gibco, Cat#PHC3015), CD3/CD28 beads (3:1 ratio beads:cells, Dynabeads™ Human T-Expander CD3/CD28 beads, Cat#11141D), prostratin (3 µmol/l, Sigma Cat#P0077-1 mg), PMA (50 ng/ml, Fisher Scientific, Cat#BP685-1) with/without Ionomycin (500 ng/ml, Tocris Biosciences, Cat#1704/1). A DMSO control was included with 0.5% v/v DMSO dissolved in RPMI media with 10% FBS. Seventy-two hours post-treatment, the ability of these constructs to induce latent activation was assessed by FACS with 10000 single events collected, and the data was analyzed using FlowJo vX3.05470 software.
NF-κB site specificity of ZFP-362-VPR: To test the target site specificity of the pcDNA-ZFP-362-VPR to activate the LTR of HIV, 100,000 pMo-HEK and pΔ362 reporter cells were seeded per well in a 24 well plate at 24 hours prior to transfection. Cells were transfected in triplicate using Lipofectamine 2000 (Thermo Fisher Scientific, MA, USA) and 72 hours post transfection, total cellular RNA was isolated from cells using the Maxwell RSC simplyRNA Cells Kit (Promega, WI, USA) according to manufacturer’s instructions. The concentrations of the isolated DNase-treated RNA samples were standardized and then reverse transcribed using the Mu-MLV Reverse Transcriptase (Thermo Fisher Scientific, MA, USA) with a n-oligo-dT/random nonamer primer mix (IDT, IA, USA).Quantitative real-time PCR (qRT-PCR) was carried out using Kapa Sybr Fast universal qPCR mix (Kapa Biosystems, MA, USA) on a Roche LightCycler® 96 System (Roche Diagnostics Corporation, IN, USA) according to provided instructions. The following PCR primer sets were used - GFP Fwd Primer: 5′-GACAACCACTACCTGAGCAC-3, GFP Rev Primer:5′-CAGGACCATGTGATCGCG-3, RPLP0 Fwd Primer: 5′-CGCAGCCAATAGACAGGAG-3, RPLP0 Rev Primer: 5′-GCGCGTGCCTTTTATAATGC-3 [53], and the GFP expression was analyzed using quantitative reverse transcription PCR (qRT-PCR). pcDNA-ZFP-VEGF-VPR and pcDNA3.1 were included as negative controls. The qPCR data was analyzed using LightCycler® software version 1.1.1320.
Activation of HIV LTR in CD4+ T-cell latency model
Naïve cells were isolated and activated by anti-IL12 (Peprotech, Cat#500-P154G), anti-IL4 (Peprotech, Cat#500-P24), TGFbeta (Peprotech 100 − 21) and anti-CD3/CD28 beads (Invitrogen, Cat#11131D). Activated cells were infected by pseudotyped pNL4.3-deltaEnv-nLuc-VSVG viruses. At Day 17, CD4+ cells were isolated using Dynabeads™ CD4 positive isolation kit (Invitrogen, Cat#11331D). At Day 18, CD4+ cells were treated with 10 ng/ml PMA (Sigma, Cat#P1585) or anti-CD3CD28 beads or transfected by plasmids (GFP, ZFP-362-VPR, or ZFP-VEGF-VPR) using by Neon electroporation (Invitrogen, Cat#mpk1096). After 72 hrs, part of the cells was stained with fixable viability dye efluor™ 450 (eBioscience, Cat#65-0863-14), anti-CD4 (Invitrogen, Cat#MHCD0405) and anti-P24 (BD, Cat#556027) antibodies, and measured by flow cytometry. The cells were counted and samples containing 2000 live cells were lysed and measured using the Nano-Glo® Luciferase Assay System (Promega, Cat#N1120).
ChIP analysis of ZFP-362-VPR
The ZFP-362-VPR, and dCas9-VPR + sgF2-362 plasmids were transfected in triplicate into 3 million pMo-HEK cells using calcium phosphate transfection. ChIP analysis was carried out on the transfected pMo-HEK cells for the myc-tagged activating complexes using anti-myc antibodies (Cell Signaling Technology, Danvers, MA). The ChIP protocol was performed 72 hours post-transfection using previously described protocols [10, 54, 55]. The ChIP elutes were purified using the MinElute PCR purification kit (Qiagen, Hidlen, Germany) and analyzed by qPCR. Relative enrichment at the LTR was determined using LTR specific primers that were described in Saayman et al.[54].
ZFP-362-VPR and dCas9-VPR + F2gRNA effects on global RNA expression
To determine the extent to which ZFP-362-VPR and dCas9-VPR + sgF2-362 disrupted RNA expression, we compared pMo-HEK cells transfected with either construct to those transfected with a pcDNA3.1 control. The pMo-HEK were seeded at 100000 cells per well in a 24-well plate and transfected with the ZFP-362-VPR, and dCas9-VPR + sgF2-362 plasmids in quadruplicate using calcium phosphate transfection. Total cellular RNA was isolated 48 hrs post-transfection from cells using the Maxwell RC simplyRNA Cells Kit (Promega, WI, USA). Sequencing was performed by Genewiz® on an Illumina platform with paired end 150 bp reads. Sequence data are available via Gene Expression Omnibus accession number GSE150382. Raw sequencing reads were processed with the nf-core [56] RNASeq pipeline version 1.4. Briefly, trimmed reads were mapped using Spliced Transcripts Alignment to a Reference (STAR) [57] to the GRCh37 reference using the ENSEMBL (release 75) annotation. Each library was subjected to extensive quality control, including estimation of library complexity, gene body coverage, and duplication rates, among other metrics detailed in the pipeline repository. Reads were counted across genomic features using Subread featureCounts [58] and merged into a matrix of counts per gene for each sample. Differential expression analysis was performed with DESeq2 [59] as implemented in the SARTools package [60]. Only genes with at least 1 read count in 1 sample were retained for the analysis. Multiple testing was corrected using the Benjamini-Hochberg method, where p-values are adjusted to control for false discovery. An adjusted p-value threshold of 0.05 was used to select differentially expressed genes. Gene enrichment analyses were performed with ClusterProfiler [61] using the full set of genes with at least 1 count in any sample as background and differentially expressed genes as the input list. Enriched GO terms or enriched KEGG pathways were selected using an FDR threshold of 0.05 with the enrichGO function of ClusterProfiler.