Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	C).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Experimental flow for microscopy-base analysis of cell morphology and formation of F-actin rich structures.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	The figure was generated by Biorender. (
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	D).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Representative confocal images of F-actin organization for control and CYTOR KD Jurkat cells after contact with anti-CD3/28 coated surfaces.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Cells were stained with fluorescent phalloidin and DAPI to visualize F-actin and cell nuclei.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Shown are merged images of both channels, scale bar = 10 μM). (
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	E).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	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). (
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	F).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Relative CYTOR RNA levels in CYTOR KD Jurkat cells relative to control cells of the cells analyzed in (E). (
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	G).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Representative images of the different morphotypes observed for Jurkat cells after anti-CD3/28 surface stimulation (analyzed as in D), (H).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	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). **
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	0.05≤p≤0.1. (
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	I).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Inhibition of actin remodeling disrupts HIV gene expression upon T cell activation.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	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.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Cells were harvested 24 hours later, and the percentage of cells expressing HIV GFP was monitored by FACS.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Data are presented as fold of activation relative to untreated cells and activated with the indicated T cell activator.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Statistical significance is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	p≤0.05. **
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	0.05≤p≤0.1.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	T cell activation elicits complex and highly dynamic signaling cascades that ultimately lead to the activation of transcription factors, including NF-κB and NF-AT, to increase the expression of T cell receptor target genes .
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	The involvement of these transcription factors in HIV gene expression, at least in part, explains the beneficial effects of T cell activation on HIV gene expression .
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Since many of the downstream signaling events elicited by TCR engagement depend on the immediate polymerization of cortical actin, we tested if CYTOR affects the actin polymerization response to TCR engagement of Jurkat T cells.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Scramble control or CYTOR KD Jurkat cells were placed on a cell stimulatory surface coated with anti-CD3/CD28 antibodies, fixed, and stained for F-actin.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Control cells displayed the typical cell spreading and formation of circumferential F-actin-rich rings (actin ring; AR) (Fig 6C, 6D, 6E and 6F).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Although CYTOR expression was only moderately reduced in KD cells (Fig 6F), fewer cells responded to TCR stimulation (approx.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	40% less cells with AR in CYTOR KD than in control cells; Fig 6E).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Detailed analysis of the different cell morphologies revealed that the CYTOR KD particularly resulted in a significant increase in the fraction of cells that were unable to both spread as well polymerize actin into an F-actin ring in response to T cell activation.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	In contrast, the frequency of cells that failed to spread despite efficient actin polymerization was unaffected (Fig 6H).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Increasing CYTOR levels by overexpression did not further increase the frequency of cells that formed ARs, did not alter the morphology of F-actin structures formed in response to TCR activation, and did not result in the formation of ARs in the absence of TCR stimulation (S5 Fig).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	We conclude that CYTOR is an important regulator of TCR-induced actin polymerization in CD4+ T cells, but its normal endogenous expression levels are not limiting for this response.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	To assess whether TCR-induced actin remodeling affects HIV gene expression in our experimental system, we measured the induction of HIV gene expression by TCR engagement in 2D10 cells, a CD4+ T cell line that carries a latent GFP cassette under the control of the LTR promoter.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Experiments were performed in the absence or presence of the actin polymerization inhibitor, Latrunculin B—an inhibitor that interferes with actin polymerization and is reversible upon washout (Fig 6I).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Stimulation with anti-CD3/anti-CD28 resulted in a marked induction of GFP expression and, as observed before, silencing CYTOR expression reduced this induction.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Notably, interfering with actin polymerization during the first 3 hours of TCR stimulation in control cells limited the induction of HIV gene expression to the levels observed upon CYTOR KD, and interference with actin dynamics in CYTOR KD did not result in an additional reduction of GFP expression.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Finally, overexpressing CYTOR rendered the TCR-mediated induction of GFP expression insensitive to Latrunculin B (Fig 6I) .
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Together, these results reveal that the regulation of host cell actin dynamics is necessary but not sufficient for the regulation of gene expression of latent HIV.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	In search of regulators of HIV latency, we profiled changes in the expression of ncRNAs by employing RNA-Seq analysis in resting and stimulated HIV-infected J-Lat 6.3 T cells, comparing RNA expression levels in cells that carry active HIV (GFP+) or latent HIV (GFP-).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Our analysis show that different transcriptional profiles exist in cells where HIV is activated versus cells where it remains latent.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	CYTOR lncRNA was identified as one of these RNAs, and its expression is elevated upon T cell stimulation, where HIV is active.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	These observations were further confirmed in primary CD4+ T cells (Fig 1).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Functional analyses show that following T cell stimulation, over-expression of CYTOR activates HIV gene expression, while its depletion inhibits viral gene expression.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Significantly, upon T cell stimulation, depletion of CYTOR promoted entry of HIV into a latent state, while its over-expression delayed entry into latency and enhanced latency reversal (Fig 2).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Effects of CYTOR on HIV infection and latency establishment were also confirmed in stimulated primary CD4+ T cells (Fig 3).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	We are aware that the model of stimulated CD4+ primary cells does not recapitulate the actual state of the reservoir, which is mainly comprise of resting CD4+ T cells that do not support HIV infection.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	As this is a limitation of the current study, we are trying to adopt a recently developed gene editing approach to lncRNAs to deplete CYTOR in this unique cell population and monitor the effects of latency kinetics without altering its activation .
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Mechanistically, our observations show that CYTOR directly binds to the HIV promoter and enhances the phosphorylation of the Ser2 CTD of RNAPII through association with P-TEFb to activate viral gene expression (Figs 4 and 5).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Changes in histone activation marks around the viral promoter in CYTOR-depleted cells also imply that CYTOR activates the proviral gene expression (Fig 4).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	In addition to the direct effects of CYTOR on HIV gene expression, we also demonstrate that CYTOR controls global gene expression.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	CYTOR is recruited to other gene promoters that are regulated by P-TEFb, like myc, NF-κB, and IL2Ra (S3 Fig).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Among the identified enriched pathways that potentially are regulated by CYTOR are those that are involved in actin dynamics.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Consistently, reduced levels of CYTOR expression are associated with reduced polymerization of cortical actin in response to TCR engagement (Fig 6).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	In turn, elevated levels of CYTOR do not further increase actin polymerization in response to T cell stimulation and cannot induce morphological responses of T cells in the absence of stimulation (S5 Fig).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Thus, CYTOR is an important regulator of TCR-induced actin polymerization in T cells.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	However, its normal endogenous expression levels are sufficient for a proper response.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	To test a mechanistic link between actin remodeling, CYTOR levels, and HIV gene expression, we inhibited actin dynamics with specific inhibitors (Fig 6I).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Effects of inhibition of actin polymerization phenocopied the effect of CYTOR depletion on HIV gene expression, suggesting that CYTOR may affect HIV gene expression by the regulation of genes that control cellular actin dynamics (Fig 6I).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Accordingly, we propose a model where CYTOR exerts its effects on global gene expression and promotes HIV gene expression by both direct and indirect effects (Fig 7).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	CYTOR directly binds the HIV promoter and recruits the elongation transcription machinery to enhance RNAPII CTD phosphorylation and deposition of active histone markers around the HIV promoter, ultimately activating HIV gene expression.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Indirectly, CYTOR controls gene targets that regulate actin dynamics in the nucleus and at the plasma membrane to optimize the response to T cell activation, presumably via the regulation of cellular gene expression.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Following T cell activation, levels of CYTOR are elevated in the nucleus.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	CYTOR is recruited to the HIV promoter and binds to P-TEFb, leading to the activation of viral gene expression.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Cellular genes regulated by CYTOR include actin remodeling genes that promote actin polymerization and the indirect activation of HIV gene expression.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Like CYTOR, other lncRNAs have been reported to occupy the HIV promoter and modulate its activity at either transcriptional or posttranscriptional levels .
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Most act as scaffolds that associate with other transcriptional activators or repressors to control HIV gene expression [35–39,58–60].
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	In the case of CYTOR, its effects on gene expression occur by recruiting the transcription elongation machinery to activate gene expression, either from the viral promoter or other cellular promoters.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	It will be essential to identify other partners that are associated with CYTOR lncRNA and control HIV promoter activity.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	As we also aim to dissect the role of CYTOR in gene expression control, specifically for HIV gene regulation, it will be essential to define how events within the nucleus are regulated by CYTOR and translated to the control of downstream effector functions of stimulated T cells.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Future studies will further identify the downstream targets of CYTOR that control actin dynamics upon T-cell activation.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	As additional pathways were identified by our RNA-seq analysis in CYTOR-depleted cells, we visualize that future work will identify novel downstream targets of CYTOR and elucidate their mechanisms of function in regulating HIV gene expression and latency.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	These may open new ways for developing novel therapeutic tools that will be integrated or substitute current strategies to successfully eliminate the HIV reservoir.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Jurkat J-Lat 6.3 T cells are immortalized human T lymphocytes that serve as a model for studying HIV latency, as it harbors a transcriptionally silent integrated HIV provirus that encodes for a GFP reporter instead of Nef, which reactivated following T cell stimulation.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	2D10 cells are also Jurkat-based T cells, carrying a mini—HIV cassette coding for Tat and rev and a 2dGFP reporter gene.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Jurkat T cells were maintained in RMPI medium (GIBCO) containing with 10% fetal bovine serum (FBS), 2mg/ml L-glutamine, penicillin-streptomycin, and non-essential amino acids (Sigma, M7145).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Cells were cultured at 37°C with 5% CO2.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Human Embryonic Kidney HEK293T, this cell line was mainly used for the production of viral-like particles were maintained in DMEM complete medium (GIBCO).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Cells were cultured at 37°C with 5% CO2.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	For the isolation of primary human CD4+ T cells, human Buffy Coats from anonymous healthy donors were obtained from the Soroka Medical Center Hospital Blood Bank.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	At day 0, PBMCs were isolated over a Ficoll gradient (Millipore).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	PBMCs were maintained at 2 x10 cells/ml overnight at 37°C.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	CD4+ T cells were isolated by negative selection with the RosetteSep Human CD4+ T Cell Enrichment Cocktail Stemcell Technologies), resulting in homogenous populations of CD4+ T cells with a purity of 90–95% as assured by flow cytometry.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	CD4+ T cells were cultured in complete RPMI media containing recombinant human IL2 at 20 U/ml (Roche) to a final concentration 10 cells/ml.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Cells were then stimulated with anti-CD3/CD28 dynabeads (Invitrogen) and further cultured for 48 hour.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	The level of activation was monitored by FACS measuring staining with APC anti human CD25 (Biolegend #302609) and Pacific Blue anti-human CD69 (Biolegend #310919).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	At day two, stimulated cells were counted, centrifuged for 5 minutes at 1500 rpm, and resuspended in fresh RPMI to a final concentration 0.5x10 cell/ml and IL-2 before transduction with high titter HIV carrying CYTOR shRNA at an MOI of 10.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	24 hr later (day three), cells were further transduced with HIVGKO lentivirus at MOI of 10.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Transduced cells were cultured in complete RPMI media containing recombinant human IL2 and dynabeads at a ratio of 25 μl human beads per 10 million cells and analyzed by FACS at day five.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	For the IP of P-TEFb, we used the following antibodies: anti-CDK9 (Abcam ab6544) or anti-CYCLIN T1 antibodies (Abcam; ab176702).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	For ChIP-qPCR for the detection of histone marks activation markers, we used anti-H3K27Ac (ab4729) and anti-H3K4me3 (ab8580).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	For detecting the different states of the phosphorylation of RNAPII CTD, we used the phosphorylated serine 2 antibody (Ser2P; ab238146) and phosphorylated serine 5 (Ser5P; ab5131).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	To monitor T cell activation following stimulation, the following antibodies were used: APC anti human CD25 (Biolegend; 302609); Pacific Blue anti human CD69 (Biolegend; 310919).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Actin remodeling in response to T cell receptor (TCR) engagement was monitored by forming circumferential F-actin rings as previously described .
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	In brief, stimulatory coverslips were prepared by coating with a 0.01% poly-L-lysine (PLL; Sigma) solution for 10 minutes at room temperature, followed by wet-chamber incubation for 3 hours at 37°C with 7 μg/ml anti-CD3 antibody (50 μl per coverslip, clone HIT3a against CD3E; BD Biosciences) in phosphate-buffered saline (PBS).
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Stimulatory coverslips were subsequently washed in PBS and stored at 4°C in PBS until use.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	5x10 cells per anti-CD3-coated coverslip, respectively) were used to seed coverslips for 4 minutes to allow TCR-mediated actin ring formation.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Cells were subsequently fixed in 3% paraformaldehyde for 15 minutes, permeabilized for 2 minutes in 0.1% TritonX-100, and blocked for 30 minutes in 1% Fetal Calf Serum (FCS) in PBS.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	F-actin was visualized with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (1:1,000, 1 hour, room temperature; Sigma).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

C).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Experimental flow for microscopy-base analysis of cell morphology and formation of F-actin rich structures.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

The figure was generated by Biorender. (

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

D).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Representative confocal images of F-actin organization for control and CYTOR KD Jurkat cells after contact with anti-CD3/28 coated surfaces.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Cells were stained with fluorescent phalloidin and DAPI to visualize F-actin and cell nuclei.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Shown are merged images of both channels, scale bar = 10 μM). (

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

E).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

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). (

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

F).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Relative CYTOR RNA levels in CYTOR KD Jurkat cells relative to control cells of the cells analyzed in (E). (

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

G).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Representative images of the different morphotypes observed for Jurkat cells after anti-CD3/28 surface stimulation (analyzed as in D), (H).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

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). **

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

0.05≤p≤0.1. (

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

I).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Inhibition of actin remodeling disrupts HIV gene expression upon T cell activation.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

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.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Cells were harvested 24 hours later, and the percentage of cells expressing HIV GFP was monitored by FACS.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Data are presented as fold of activation relative to untreated cells and activated with the indicated T cell activator.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Statistical significance is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

p≤0.05. **

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

0.05≤p≤0.1.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

T cell activation elicits complex and highly dynamic signaling cascades that ultimately lead to the activation of transcription factors, including NF-κB and NF-AT, to increase the expression of T cell receptor target genes .

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

The involvement of these transcription factors in HIV gene expression, at least in part, explains the beneficial effects of T cell activation on HIV gene expression .

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Since many of the downstream signaling events elicited by TCR engagement depend on the immediate polymerization of cortical actin, we tested if CYTOR affects the actin polymerization response to TCR engagement of Jurkat T cells.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Scramble control or CYTOR KD Jurkat cells were placed on a cell stimulatory surface coated with anti-CD3/CD28 antibodies, fixed, and stained for F-actin.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Control cells displayed the typical cell spreading and formation of circumferential F-actin-rich rings (actin ring; AR) (Fig 6C, 6D, 6E and 6F).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Although CYTOR expression was only moderately reduced in KD cells (Fig 6F), fewer cells responded to TCR stimulation (approx.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

40% less cells with AR in CYTOR KD than in control cells; Fig 6E).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Detailed analysis of the different cell morphologies revealed that the CYTOR KD particularly resulted in a significant increase in the fraction of cells that were unable to both spread as well polymerize actin into an F-actin ring in response to T cell activation.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

In contrast, the frequency of cells that failed to spread despite efficient actin polymerization was unaffected (Fig 6H).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Increasing CYTOR levels by overexpression did not further increase the frequency of cells that formed ARs, did not alter the morphology of F-actin structures formed in response to TCR activation, and did not result in the formation of ARs in the absence of TCR stimulation (S5 Fig).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

We conclude that CYTOR is an important regulator of TCR-induced actin polymerization in CD4+ T cells, but its normal endogenous expression levels are not limiting for this response.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

To assess whether TCR-induced actin remodeling affects HIV gene expression in our experimental system, we measured the induction of HIV gene expression by TCR engagement in 2D10 cells, a CD4+ T cell line that carries a latent GFP cassette under the control of the LTR promoter.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Experiments were performed in the absence or presence of the actin polymerization inhibitor, Latrunculin B—an inhibitor that interferes with actin polymerization and is reversible upon washout (Fig 6I).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Stimulation with anti-CD3/anti-CD28 resulted in a marked induction of GFP expression and, as observed before, silencing CYTOR expression reduced this induction.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Notably, interfering with actin polymerization during the first 3 hours of TCR stimulation in control cells limited the induction of HIV gene expression to the levels observed upon CYTOR KD, and interference with actin dynamics in CYTOR KD did not result in an additional reduction of GFP expression.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Finally, overexpressing CYTOR rendered the TCR-mediated induction of GFP expression insensitive to Latrunculin B (Fig 6I) .

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Together, these results reveal that the regulation of host cell actin dynamics is necessary but not sufficient for the regulation of gene expression of latent HIV.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

In search of regulators of HIV latency, we profiled changes in the expression of ncRNAs by employing RNA-Seq analysis in resting and stimulated HIV-infected J-Lat 6.3 T cells, comparing RNA expression levels in cells that carry active HIV (GFP+) or latent HIV (GFP-).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Our analysis show that different transcriptional profiles exist in cells where HIV is activated versus cells where it remains latent.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

CYTOR lncRNA was identified as one of these RNAs, and its expression is elevated upon T cell stimulation, where HIV is active.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

These observations were further confirmed in primary CD4+ T cells (Fig 1).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Functional analyses show that following T cell stimulation, over-expression of CYTOR activates HIV gene expression, while its depletion inhibits viral gene expression.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Significantly, upon T cell stimulation, depletion of CYTOR promoted entry of HIV into a latent state, while its over-expression delayed entry into latency and enhanced latency reversal (Fig 2).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Effects of CYTOR on HIV infection and latency establishment were also confirmed in stimulated primary CD4+ T cells (Fig 3).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

We are aware that the model of stimulated CD4+ primary cells does not recapitulate the actual state of the reservoir, which is mainly comprise of resting CD4+ T cells that do not support HIV infection.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

As this is a limitation of the current study, we are trying to adopt a recently developed gene editing approach to lncRNAs to deplete CYTOR in this unique cell population and monitor the effects of latency kinetics without altering its activation .

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Mechanistically, our observations show that CYTOR directly binds to the HIV promoter and enhances the phosphorylation of the Ser2 CTD of RNAPII through association with P-TEFb to activate viral gene expression (Figs 4 and 5).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Changes in histone activation marks around the viral promoter in CYTOR-depleted cells also imply that CYTOR activates the proviral gene expression (Fig 4).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

In addition to the direct effects of CYTOR on HIV gene expression, we also demonstrate that CYTOR controls global gene expression.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

CYTOR is recruited to other gene promoters that are regulated by P-TEFb, like myc, NF-κB, and IL2Ra (S3 Fig).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Among the identified enriched pathways that potentially are regulated by CYTOR are those that are involved in actin dynamics.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Consistently, reduced levels of CYTOR expression are associated with reduced polymerization of cortical actin in response to TCR engagement (Fig 6).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

In turn, elevated levels of CYTOR do not further increase actin polymerization in response to T cell stimulation and cannot induce morphological responses of T cells in the absence of stimulation (S5 Fig).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Thus, CYTOR is an important regulator of TCR-induced actin polymerization in T cells.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

However, its normal endogenous expression levels are sufficient for a proper response.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

To test a mechanistic link between actin remodeling, CYTOR levels, and HIV gene expression, we inhibited actin dynamics with specific inhibitors (Fig 6I).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Effects of inhibition of actin polymerization phenocopied the effect of CYTOR depletion on HIV gene expression, suggesting that CYTOR may affect HIV gene expression by the regulation of genes that control cellular actin dynamics (Fig 6I).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Accordingly, we propose a model where CYTOR exerts its effects on global gene expression and promotes HIV gene expression by both direct and indirect effects (Fig 7).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

CYTOR directly binds the HIV promoter and recruits the elongation transcription machinery to enhance RNAPII CTD phosphorylation and deposition of active histone markers around the HIV promoter, ultimately activating HIV gene expression.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Indirectly, CYTOR controls gene targets that regulate actin dynamics in the nucleus and at the plasma membrane to optimize the response to T cell activation, presumably via the regulation of cellular gene expression.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Following T cell activation, levels of CYTOR are elevated in the nucleus.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

CYTOR is recruited to the HIV promoter and binds to P-TEFb, leading to the activation of viral gene expression.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Cellular genes regulated by CYTOR include actin remodeling genes that promote actin polymerization and the indirect activation of HIV gene expression.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Like CYTOR, other lncRNAs have been reported to occupy the HIV promoter and modulate its activity at either transcriptional or posttranscriptional levels .

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Most act as scaffolds that associate with other transcriptional activators or repressors to control HIV gene expression [35–39,58–60].

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

In the case of CYTOR, its effects on gene expression occur by recruiting the transcription elongation machinery to activate gene expression, either from the viral promoter or other cellular promoters.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

It will be essential to identify other partners that are associated with CYTOR lncRNA and control HIV promoter activity.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

As we also aim to dissect the role of CYTOR in gene expression control, specifically for HIV gene regulation, it will be essential to define how events within the nucleus are regulated by CYTOR and translated to the control of downstream effector functions of stimulated T cells.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Future studies will further identify the downstream targets of CYTOR that control actin dynamics upon T-cell activation.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

As additional pathways were identified by our RNA-seq analysis in CYTOR-depleted cells, we visualize that future work will identify novel downstream targets of CYTOR and elucidate their mechanisms of function in regulating HIV gene expression and latency.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

These may open new ways for developing novel therapeutic tools that will be integrated or substitute current strategies to successfully eliminate the HIV reservoir.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Jurkat J-Lat 6.3 T cells are immortalized human T lymphocytes that serve as a model for studying HIV latency, as it harbors a transcriptionally silent integrated HIV provirus that encodes for a GFP reporter instead of Nef, which reactivated following T cell stimulation.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

2D10 cells are also Jurkat-based T cells, carrying a mini—HIV cassette coding for Tat and rev and a 2dGFP reporter gene.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Jurkat T cells were maintained in RMPI medium (GIBCO) containing with 10% fetal bovine serum (FBS), 2mg/ml L-glutamine, penicillin-streptomycin, and non-essential amino acids (Sigma, M7145).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Cells were cultured at 37°C with 5% CO2.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Human Embryonic Kidney HEK293T, this cell line was mainly used for the production of viral-like particles were maintained in DMEM complete medium (GIBCO).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Cells were cultured at 37°C with 5% CO2.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

For the isolation of primary human CD4+ T cells, human Buffy Coats from anonymous healthy donors were obtained from the Soroka Medical Center Hospital Blood Bank.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

At day 0, PBMCs were isolated over a Ficoll gradient (Millipore).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

PBMCs were maintained at 2 x10 cells/ml overnight at 37°C.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

CD4+ T cells were isolated by negative selection with the RosetteSep Human CD4+ T Cell Enrichment Cocktail Stemcell Technologies), resulting in homogenous populations of CD4+ T cells with a purity of 90–95% as assured by flow cytometry.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

CD4+ T cells were cultured in complete RPMI media containing recombinant human IL2 at 20 U/ml (Roche) to a final concentration 10 cells/ml.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Cells were then stimulated with anti-CD3/CD28 dynabeads (Invitrogen) and further cultured for 48 hour.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

The level of activation was monitored by FACS measuring staining with APC anti human CD25 (Biolegend #302609) and Pacific Blue anti-human CD69 (Biolegend #310919).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

At day two, stimulated cells were counted, centrifuged for 5 minutes at 1500 rpm, and resuspended in fresh RPMI to a final concentration 0.5x10 cell/ml and IL-2 before transduction with high titter HIV carrying CYTOR shRNA at an MOI of 10.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

24 hr later (day three), cells were further transduced with HIVGKO lentivirus at MOI of 10.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Transduced cells were cultured in complete RPMI media containing recombinant human IL2 and dynabeads at a ratio of 25 μl human beads per 10 million cells and analyzed by FACS at day five.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

For the IP of P-TEFb, we used the following antibodies: anti-CDK9 (Abcam ab6544) or anti-CYCLIN T1 antibodies (Abcam; ab176702).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

For ChIP-qPCR for the detection of histone marks activation markers, we used anti-H3K27Ac (ab4729) and anti-H3K4me3 (ab8580).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

For detecting the different states of the phosphorylation of RNAPII CTD, we used the phosphorylated serine 2 antibody (Ser2P; ab238146) and phosphorylated serine 5 (Ser5P; ab5131).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

To monitor T cell activation following stimulation, the following antibodies were used: APC anti human CD25 (Biolegend; 302609); Pacific Blue anti human CD69 (Biolegend; 310919).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Actin remodeling in response to T cell receptor (TCR) engagement was monitored by forming circumferential F-actin rings as previously described .

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

In brief, stimulatory coverslips were prepared by coating with a 0.01% poly-L-lysine (PLL; Sigma) solution for 10 minutes at room temperature, followed by wet-chamber incubation for 3 hours at 37°C with 7 μg/ml anti-CD3 antibody (50 μl per coverslip, clone HIT3a against CD3E; BD Biosciences) in phosphate-buffered saline (PBS).

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Stimulatory coverslips were subsequently washed in PBS and stored at 4°C in PBS until use.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

5x10 cells per anti-CD3-coated coverslip, respectively) were used to seed coverslips for 4 minutes to allow TCR-mediated actin ring formation.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Cells were subsequently fixed in 3% paraformaldehyde for 15 minutes, permeabilized for 2 minutes in 0.1% TritonX-100, and blocked for 30 minutes in 1% Fetal Calf Serum (FCS) in PBS.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

F-actin was visualized with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (1:1,000, 1 hour, room temperature; Sigma).