Fig 1.
Identifying host ncRNAs that are differentially expressed in Jurkat T cells that carry active or latent HIV following T-cell activation.
(A) FACS histogram analysis of PMA/Ionomycin (P/I)-stimulated J-Lat 6.3 cells. GFP(+) cells carrying active HIV (P1 region) were sorted from GFP (-) cells carrying latent HIV (GFP-). Cells were sorted and sent to RNA-Seq (n = 4). (B) Heatmap of differential transcript expression pattern (FC≥ a 2-fold change and above between cells carrying active versus latent HIV with an adjusted p value of ≤0.05. (C) Pie chart corresponding to the numbers of differentially expressed mRNAs and lncRNAs, up and downregulated in cells where HIV was reactivated. (D) RNA levels of selected ncRNA in primary CD4+ T cells. Analysis of expression levels of selected ncRNA based on the RNA-Seq analysis in primary CD4+ T cells that were either under resting conditions (-) or stimulated with P/I (+). RNA levels were analyzed by RT-qPCR. Data were normalized to gapdh levels. Data are from 2 healthy donors.
Fig 2.
Effects of CYTOR expression on HIV gene expression and latency establishment.
(A). Modulation of CYTOR RNA levels in J-Lat 6.3 cells. RT-qPCR analysis measuring CYTOR RNA levels in J-Lat 6.3 T cells, where CYTOR expression is knockdown (KD; red bar) or overexpressed (light blue bar). RNA levels were normalized to gapdh and presented relative to control cells expressing scrambled shRNA (grey bar). Statistical significance is based on calculating ±SD of data points from four independent experiments using two-way ANOVA. ***p≤0.05. (B) Effects of CYTOR on HIV gene expression. FACS quantification analysis of the percentage of cells that express HIV-GFP in P/I stimulated J-Lat 6.3 cells expressing control scramble shRNA (grey bar), or in which CYTOR was overexpressed (blue bar) or knockdown (KD; red bar). Statistical significance is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***p≤0.05. (C) Kinetics of latency establishment in the context of CYTOR expression. 2D10 latency model Jurkat T cells that carry a mini-Tat-Rev GFP under the regulation of the HIV LTR promoter and express either scrambled shRNA (grey), CYTOR KD (red), or cells over-expressing CYTOR (blue) were reactivated and sorted to obtain a pure cell population that expresses GFP. GFP expression was then followed over time as a measurement of entry into latency. Statistical significance is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***p≤0.05 and ns: not significant.
Fig 3.
CYTOR depletion in primary CD4+ T cells suppresses HIV infection and promotes latency establishment.
(A). Experimental workflow overview for isolating primary CD4+ T cells. See methods for a detailed description. The figure was generated by Biorender. (B). Depletion of CYTOR in stimulated primary CD4+ T cells using lentivirus encoding CYTOR shRNA. Data were measured by RT-qPCR, normalized to GAPDH, and presented relative to cells expressing scrambled shRNA—set to 1. Statistical significance is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***p≤0.05; n = 3. (C). FACS analysis presenting effects of CYTOR knockdown (KD) on HIVGKO infection in primary CD4+ T cells. Cells were stimulated and then transduced with HIVGKO before being analyzed by FACS for mKO2 and GFP expression. (D). Quantification of quadrate percentage from three independent experiments of FACS analysis for HIVGKO transduction in CD4+ primary T cells, where CYTOR is KD. Statistical significance is based on the calculation of mean ±SD from three independent experiments (n = 3) using Two-way ANOVA. ***p≤0.05.
Fig 4.
CYTOR is localized in the nucleus, binds to the HIV promoter, and modifies RNAPII CTD phosphorylation state and histone landscape.
(A). CYTOR is localized to the nucleus and its levels are elevated upon T-cell stimulation. Resting or stimulated primary CD4+ T cells were subjected to cell fractionation, separating the samples into a nuclear fraction (grey bar) or cytoplasmic fraction (light blue bar). Samples were then subjected to RT-qPCR and monitored for CYTOR or 7SK ncRNA levels. Data were normalized to 7SL RNA in each of the cellular fractions and conditions. Data are presented relative to cytoplasmic fraction in each condition—set to 1. (B). ChIRP-qPCR analysis for CYTOR binding to the HIV promoter. CYTOR-specific (black bar) or control lacZ (red bar) antisense biotinylated probes were incubated with lysates isolated from unstimulated or P/I stimulated J-Lat 6.3 cells. Biotinylated RNA was pulled down with streptavidin beads, and following washing, associated DNA was eluted and analyzed by qPCR with primers for the HIV promoter. Statistical significance was calculated between the two probes and between unstimulated and stimulated states. IgG served as a non-specific antibody for IP control (grey bar). The analysis is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***p≤0.05. **0.05≤p≤ 0.1; n.s—not significant. (C, D) CYTOR affects the phosphorylation state of RNAPII CTD and histone landscape. ChIP qPCR analysis in control or CYTOR KD J-Lat 6.3 cells. ChIP material from cells was immune-precipitated (IP) with antibodies targeting RNAPII-Ser2P or RNAPII Ser5P (C); or for H3K4me3 and H3K27Ac histone activation marks (D). IP fraction was analyzed for enrichment of the indicated modifications on the HIV promoter by qPCR with specific primers. Non-specific IgG served as a control (grey bar). Percentage of input are means ±SD; n = 3; *** p≤0.05 calculated between scrambled and KD cells for each antibody. n.s—not significant.
Fig 5.
CYTOR associates with P-TEFb in cells.
(A) RIP analysis demonstrates the association of CYTOR with P-TEFb. Isolated ChIP material from resting or stimulated J-Lat 6.3 CD4+ T cells was subjected to immune precipitation with antibodies targeting CDK9 or CYCLIN T1 of P-TEFb, followed by RT-qPCR with primers for the relevant lncRNA (7SK or CYTOR). Non-specific IgG served as a control for the IP step. 7SL ncRNA served as a control for an RNA that does not associate with P-TEFb and, therefore, not precipitated with CDK9 or CYCLIN T1 antibodies. Statistical significance is based on the calculation of mean ±SD from three independent experiments using two-way ANOVA. ***p≤0.05. ** 0.05≤p≤0.1; ns: not significant. (B) CYTOR associates with P-TEFb in cells. RNA pull-down followed by western blotting where lysates from J-Lat 6.3 cells were incubated with an in-vitro transcribed biotinylated CYTOR anti-sense probe and reactions were pulled down with streptavidin beads. Eluted RNP complexes were subjected to western blotting with indicated antibodies. Non-specific IgG served as a control for non-relevant IgG. Scramble RNA served as RNA that does not associate with P-TEFb. 7SK probe confirmed association with P-TEFb. Input is 5% of the total cell lysate [63].
Fig 6.
CYTOR is required for TCR-induced cytoplasmic actin remodeling.
(A). Volcano plot of the expression pattern of genes from an RNA-Seq analysis upon CYTOR depletion following T cell stimulation. -Log10 P is shown on the y-axis, and Log2FC is on the x-axis. RNA was isolated from 3 biological replicates (n = 3). The fold of change cutoff is defined as FC ≥2. FDR of p≤0.05 was used as a cutoff for significance. (B). Gene Ontology analysis for enriched CYTOR gene targets. For enrichment analysis, the DAVID program was employed to identify enriched pathways and terms associated with the selected genes. (C). Experimental flow for microscopy-base analysis of cell morphology and formation of F-actin rich structures. The figure was generated by Biorender. (D). Representative confocal images of F-actin organization for control and CYTOR KD Jurkat cells after contact with anti-CD3/28 coated surfaces. Cells were stained with fluorescent phalloidin and DAPI to visualize F-actin and cell nuclei. Shown are merged images of both channels, scale bar = 10 μM). (E). Relative frequency of cells with circumferential F actin ring (AR) in control or CYTOR KD cells with proper cell spreading and circumferential F-actin relative to control cells (mean± SD, 100 cells per experiment/condition, n = 3). (F). Relative CYTOR RNA levels in CYTOR KD Jurkat cells relative to control cells of the cells analyzed in (E). (G). Representative images of the different morphotypes observed for Jurkat cells after anti-CD3/28 surface stimulation (analyzed as in D), (H). Quantifying the frequency of the morphotypes defined in (G) for control and CYTOR KD Jurkat cells (mean± SD, 100 cells per experiment/condition, n = 3). ** 0.05≤p≤0.1. (I). Inhibition of actin remodeling disrupts HIV gene expression upon T cell activation. 2D10 cells carrying an integrated HIV-GFP provirus where CYTOR expression was either depleted or over-expressed were treated with an actin polymerization inhibitor, Latrunculin B (LanB) for 1 hour, followed by T cell stimulation with anti-CD3/CD28 for an additional 3 hours. Cells were harvested 24 hours later, and the percentage of cells expressing HIV GFP was monitored by FACS. Data are presented as fold of activation relative to untreated cells and activated with the indicated T cell activator. Statistical significance is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***p≤0.05. ** 0.05≤p≤0.1.
Fig 7.
A working model for CYTOR functions.
Following T cell activation, levels of CYTOR are elevated in the nucleus. CYTOR is recruited to the HIV promoter and binds to P-TEFb, leading to the activation of viral gene expression. Cellular genes regulated by CYTOR include actin remodeling genes that promote actin polymerization and the indirect activation of HIV gene expression.