Fig 1.
Schematic of targeted CRISPR screen for novel latency regulating factors.
In this study we conduct a targeted CRISPR screen for novel latency regulating factors. HIV-1 correlated factors were first identified using a prior dataset and a 351 target pooled gRNA library (TxLatent) was constructed using the HIV-CRISPR vector that contains an HIV-1 RNA packaging signal. Screening was carried out in J-Lat 10.6, a Jurkat cell-based model of HIV-1 latency. gRNAs that reactivate or inhibit HIV-1 expression are enriched or depleted in secreted virions found in the cellular supernatant respectively. Screening was also carried out in the presence or absence of four different latency reversing agents (LRAs).
Fig 2.
CRISPR Screen of TxLatent library.
Latency HIV-CRISPR screen data is displayed for J-Lat 10.6 cells transduced with TxLatent library and subsequently treated with DMSO (A), AZD5582 (10nM) (B), prostratin (75nM) (C), iBET151 (75nM) (D), or vorinostat (500nM) (E). Genes in the TxLatent library are randomly displayed on the x-axis, in the same order across each treatment. The -Log(MAGeCK Score), a statistical pipeline that takes into account the enrichment/depletion of all guides for a given gene as well as the variation between replicates [16] is plotted for the enriched genes and the Log(MAGeCK score) is plotted for depleted genes, and combined together to display on a single graph for each treatment. A list of “synthetic” NTCs (synNTCs) was created by randomly grouping sets of 8 NTCs and used to generate a cutoff for gene hits that were enriched or depleted. SynNTCs are shown with white circles and genes targeted by gRNAs in the TxLatent library are shown with gray circles. The red lines on each graph represent the two standard deviations of mean of the synNTCs: DMSO, 1.52 and -1.91; AZD5582, 1.53 and -2.10; iBET151, 1.75 and -1.87; prostratin, 1.58 and -2.19; and vorinostat, 1.61 and -1.9.
Fig 3.
CRISPR validation of targets in HIV-1 infected primary CD4 T cells.
(A) Schematic overview of experimental design for CRISPR-Cas9 targeting of host genes in HIV-GFP infected primary CD4 T cells. Activated CD4 T cells were infected with HIV-GFP [26]. At 3 days post infection, infected (GFP+) cells were enriched by magnetic sorting and cultured for 4 days before nucleofection of Cas9/gRNA complexes targeting AAVS1, SMC3, CDK6, ZIC5, FOXE3, SAMD12, ZNF740, ETS1, and TNFAIP3 or a non-targeting gRNA (NT). Pools of 3 different gRNAs were used for each target. HIV-GFP infected cells are denoted in green color. Differential intensity of green color represents latent and active infection. (B) Relative level of HIV-1 expression measured as GFP using flow cytometry. GFP expression was measured at two weeks post nucleofection. Data are displayed as GFP expression in fold change normalized to cells nucleofected with NT gRNA. Each condition represents three biological replicates/donors (Yellow, Blue and Purple) and three technical replicates for each donor. (C) Dot plots representing viral GFP expression at two weeks post knockout. (D) Western blot of the host cellular factors targeting CDK6, ZIC5, ZNF740, ETS1, and TNFAIP3 or a control non-targeting (NT) Cas9/gRNA nucleofected primary cells at one week post nucleofection (pKO). Statistical analysis was conducted for cumulative data. Error bars represent standard deviations, and P values displayed were determined by two-way ANOVA Tukey’s multiple comparisons test. WT, wildtype; NT, non-target; CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR-associated protein 9.
Fig 4.
ETS1 knockout in primary CD4 T cells reveals positive and negative roles in HIV-1 expression.
Primary CD4 T cells were activated, infected with HIV-GFP, enriched and nucleofected as in Fig 3. (A) Western blot of the ETS1-targeting or a control non-targeting (NT) Cas9/gRNA nucleofected primary cells for beta actin and ETS1 expression at one and two weeks post nucleofection (pKO). (B-E) Flow cytometry of infected cells without stimulation or after 24 h of stimulation with one of four different LRAs (vorinostat, prostratin, iBET151, or AZD5582) at one (B) and two (D) week post nucleofection. (C and E) Dot plots representing viral GFP expression at one and two weeks post knockout respectively. Data are represented as fold change in percent GFP expression, normalized to cells nucleofected with NT gRNA. Error bars represent standard deviation, and P values displayed were determined by two-way ANOVA Tukey’s multiple comparisons test. Data shown are representative of two independent experiments with different donors, with biological triplicates for each condition.
Fig 5.
ETS1 knockout reactivates HIV-1 transcription in cell line models of HIV-1 latency and in latently infected primary CD4 T cells.
CRISPR knockout was carried out in three latently infected cell lines, J-Lat 10.6, 2D10, and Jurkat-N6 cells, and in latently infected CD4 T cells in vitro with non-targeting or ETS1 targeting gRNAs. Cells harvested at 1 week post nucleofection were quantified for ETS1 depletion and HIV-1 reactivation using western blot and flow cytometry, respectively. (A) Depletion of ETS1 in nucleofected J-Lat 10.6, 2D10, Jurkat-N6 cells, and latently infected primary CD4 T cells was analyzed by western blot. (B) Relative GFP expression in latency cell line models, and in latently infected CD4 T cells nucleofected with ETS1-targeting or NT control Cas9/gRNA complexes. GFP expression was normalized to cells nucleofected with non-target gRNA and presented as fold change. Experiment was conducted with three biological replicates for each condition. Error bars represent standard deviation, and P values displayed were determined by two-way ANOVA Tukey’s multiple comparisons test.
Fig 6.
ETS1 knockout reactivates HIV-1 transcription in CD4 T cells from people with HIV-1 (PWH) on antiretroviral therapy (ART) ex vivo.
(A) Schematic of ex vivo CRISPR nucleofection experiment in resting CD4 T cells isolated from PWH on ART. CRISPR nucleofection of ETS1-targeting or non-targeting (NT) Cas9/gRNAs was performed after a 24 h culture of PWH CD4 T cells followed by quantification of Gag RNA expression at 3 days post nucleofection by quantitative PCR. (B) Depletion of ETS1 in nucleofected resting CD4 T cells was analyzed by western blot. (C) Relative abundance of Gag viral RNA expression in CD4 T cells nucleofected with ETS1-targeting or NT control Cas9/gRNA complexes. Gag viral RNA expression was normalized to cells nucleofected with non-targeting gRNA and presented as fold change. Experiment was conducted in three biological replicates/donors with three technical replicates for each condition. Error bars represent standard deviation, and P values displayed were determined by two-way ANOVA Tukey’s multiple comparisons test. Figure elements prepared using Biorender.
Fig 7.
Analysis of differential gene expression in CD4 T cells from PWH on ART after depletion of ETS1.
CD4 T cells from three PWH on ART were nucleofected with Cas9/gRNA complexes targeting ETS1 or non-targeting control (NT) as described in Fig 6, followed by bulk RNAseq. (A) Principal component analysis (PCA) plot of samples based on gene expression data for each sample. ETS1-targeted samples shown by blue dots, non-targeting gRNA samples by red dots. Each data point represents an individual sample. The y-axis and x-axis represent the first and second principal components, respectively. (B) Volcano plot of overall differentially expressed genes (DEGs) between HIV-1 infected CD4 T cells nucleofected with ETS1 targeting sgRNA/Ca9 complexes vs non-targeting. (C) Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis of upregulated and downregulated genes of the RNA-sequencing analysis of ETS1 vs control cells. The 10 most significant KEGG pathways are shown.
Fig 8.
Reactivation of HIV-1 following ETS1 depletion is partially dependent on MALAT1.
In vitro generated primary latent CD4 T cells were nucleofected with Cas9/gRNA ribonucleoprotein complexes targeting ETS1, MALAT1, or a combination of ETS1 and MALAT1 as described in Fig 6, followed by immunoblotting, flow cytometry and RT-PCR. (A) Depletion of ETS1 in nucleofected CD4 T cells was analyzed by western blot. (B-D) Relative abundance of MALAT1 RNA expression (B), and Gag RNA (C), and HIV-1 protein expression (D) in CD4 T cells nucleofected with Cas9/sgRNAs targeting ETS1, MALAT1, or a combination of ETS1 and MALAT1. MALAT1 and Gag viral RNA expression was normalized to cells nucleofected with NT control. Experiment was conducted in three biological replicates/donors with three technical replicates for each condition. Error bars represent standard deviations, and P values displayed were determined by two-way ANOVA Tukey’s multiple comparisons test.
Fig 9.
ETS1 depletion increases the abundance of transcription activating histone marks at the HIV-1 LTR.
J-Lat 10.6 cells were nucleofected with ETS1 targeting or control non-targeting gRNAs. At 1 week post nucleofection, nucleofected cells were analyzed by Cleavage Under Targets & Release Using Nuclease (CUT&RUN) using antibodies against H3K9ac, H3K27ac and controls H3K4Me3 and IgG. (A) Experimental overview. (B) Western blot of the ETS1-targeting or control non-targeting (NT) gRNA nucleofected J-Lat10.6 cells for beta actin and ETS1 expression at one- week post nucleofection. (C) %GFP+ cells by flow cytometry 1 week post nucleofection with ETS1 and NT gRNA/Cas9 RNPs. (D) Abundance of Gag unspliced viral RNA expression in J-Lat 10.6 cells nucleofected with ETS1-targeting or NT control gRNA/Cas9 RNPs. Error bars represent standard deviation, and P values displayed were determined by two-way ANOVA Tukey’s multiple comparisons test. (E) Normalized coverage of sequencing reads (aggregated from 3 CUT&RUN technical replicates for each condition) across the HIV-1 reference genome and MALAT1 (chr11:65,498,907-65,506,539) is displayed using Integrative Genomics Viewer version 2.17. The HIV-1 LTR promoter region is denoted in a box. Figure elements prepared using Biorender.