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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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7–9].
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Alternatively, a “Block and Lock” approach frees infected individuals from ART by silencing HIV transcription and inducing a deep state of latency.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Nevertheless, despite promising therapeutic options, these strategies and others have regretfully failed to achieve significant clinical efficacy.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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These failures highlight our lack of knowledge of the molecular mechanisms that govern latency establishment and reversal and the need for alternative therapies capable of eliminating the viral reservoirs [10–15].
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Epigenetic constraints that suppress proviral gene transcription are essential for establishing HIV latency .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Low levels of basal and elongating transcription factors in the infected T cell, together with the absence of the viral trans-activator of transcription (Tat), ensure that proviral transcription remains below detectable thresholds .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Within the infected T cells, gene transcription of the integrated provirus and the host genome are synchronized .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Both display key steps of gene transcription, which include initiation, promoter arrest, and elongation.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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HIV-Tat orchestrates transcription elongation of the provirus by binding to TAR RNA and recruiting P-TEFb and Super Elongation Complex (SEC) to the viral promoter [22–26].
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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However, despite extensive efforts to elucidate the mechanisms of metazoan transcriptional control and its role in the regulation of HIV gene transcription, the knowledge of how HIV latency is established is still incomplete .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Long non-coding RNAs (lncRNAs) are transcripts with longer than 200 nucleotides that lack protein-coding capacity.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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To date, over 200,000 cell type-specific lncRNAs have been identified and display critical regulatory functions of many processes within cells [28–31].
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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However, the functions of most of these transcripts remain poorly understood.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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In the context of HIV, roles for several cellular lncRNAs have been documented [32–40].
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Moreover, significant gaps still remain in our knowledge about the mechanistic roles that lncRNAs play in CD4 T cell activation and HIV latency.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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In this study, we monitored changes in gene expression in an HIV-infected Jurkat-derived T cell line (J-Lat 6.3) upon response to T cell stimulation with Phorbol 12-myristate 13-acetate—PMA/Ionomycin (P/I).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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We documented RNA expression in stimulated J-Lat 6.3 cells that carry either active or cells latent HIV, and among identified ncRNA, Cytoskeleton Regulator RNA (CYTOR) exhibited a profound change in expression in cells that expressed active HIV following T cell stimulation.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CYTOR directly binds the HIV promoter and activates viral gene transcription and latency reversal by recruiting P-TEFb to the viral promoter.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CYTOR also exerts its effects indirectly by controlling global gene expression along with actin dynamic pathways, thereby affecting T cell activation and HIV infection.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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In search for novel host regulators of HIV gene expression and viral latency, we employed RNA-Sequencing analysis to monitor changes in the transcriptome of Jurkat-derived J-Lat 6.3.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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These cells carry a transcriptionally repressed intact copy of HIV-1 proviral DNA with a GFP reporter under the control of the HIV promoter that is inserted in the nef gene.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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As expected, in response to T-cell stimulation, HIV gene expression in J-Lat 6.3 cells was enhanced, as exhibited by elevated expression levels of GFP .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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J-Lat 6.3 cells were stimulated with the PKC activator PMA and Ionomycin (P/I), which potently activate CD4+ T lymphocytes.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Stimulated J-Lat 6.3 cells were then sorted by FACS based on their HIV-GFP expression and divided into two distinct populations: Stimulated cells that expressed HIV genes (GFP+; P1) and stimulated cells that carried latent provirus (GFP-) (Fig 1A).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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RNA from both cell groups was isolated, and libraries were generated for transcriptome analysis by next generation sequencing (RNA-Seq).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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As expected, a pronounced change in cellular gene expression, including mRNAs, miRNAs, snoRNAs, snRNAs and lncRNAs was observed in stimulated cells that expressed active HIV or latent HIV (Fig 1B).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Subsequent RNA-Seq from HIV expressing cells that carry active (GFP+) or latent (GFP-), indicated different transcriptional profiles in cells where HIV is activated versus cells where the virus remained latent.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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A total of 3490 annotated transcripts were identified whose expression was changed in cells that carried transcriptionally active HIV relative to latent HIV.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Of these, 2400 transcripts corresponded to protein-coding genes, while 843 were lncRNAs.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Upon T-cell stimulation, 468 lncRNA transcripts were upregulated (enriched in cells expressing active HIV; GFP+), and 375 were downregulated (enriched in cells carrying latent HIV provirus; GFP-) (Fig 1C).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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We further assessed the relative expression levels of the highly ranked lncRNAs in CD4+ T primary T cells by RT-qPCR.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Analysis was performed under resting or stimulating conditions of primary CD4+ T cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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For most tested lncRNAs, a shift in expression levels was confirmed when comparing primary CD4+ T cells under resting or stimulated conditions, where HIV was latent or active, respectively (Fig 1D).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Notably, ncRNAs with reported effects on HIV replication and latency, including HEAL and NRON were identified via our RNA-Seq analysis, demonstrating the potential of this screening approach (Fig 1D).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Also indicated are ncRNAs that are currently under investigation like IL21R-AS; PCBP3-AS; APOBEC3B-AS; IER3-AS (Fig 1D).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Similarly, mRNAs genes, including HSP90 , ESR-1 , and IFI16 , that were previously reported to control HIV replication were also identified by our screening approach (S1 Table; GSE254771).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Finally, we confirmed surface expression of CD25 and CD69 activation markers in stimulated primary CD4+ T cells that were infected with HIVGKO and carry active or latent HIV (S1 Fig). (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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A) FACS histogram analysis of PMA/Ionomycin (P/I)-stimulated J-Lat 6.3 cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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GFP(+) cells carrying active HIV (P1 region) were sorted from GFP (-) cells carrying latent HIV (GFP-).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Cells were sorted and sent to RNA-Seq (n = 4). (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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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. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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C) Pie chart corresponding to the numbers of differentially expressed mRNAs and lncRNAs, up and downregulated in cells where HIV was reactivated. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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D) RNA levels of selected ncRNA in primary CD4+ T cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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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 (+).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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RNA levels were analyzed by RT-qPCR.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Data were normalized to gapdh levels.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Data are from 2 healthy donors.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Among the lncRNAs that were strongly induced upon T cell stimulation, we focused our work on Cytoskeleton Regulator RNA (CYTOR)—also known as lincRNA00152.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Elevated RNA levels of CYTOR upon T cell stimulation were confirmed in J-Lat 6.3 and in primary CD4+ T cells (Fig 1D).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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To monitor the effects of HIV infection on CYTOR RNA levels, Jurkat T cells were infected with HIV and levels of CYTOR RNA were determined by RT qPCR relative to non-infected cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Our analysis confirmed that CYTOR RNA levels were not affected by HIV infection (S2 Fig).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CYTOR is an intergenic 828 nucleotide lncRNA located on chromosome 2p11.2.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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It is highly conserved in primates and rodents but less so in lower organisms.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CYTOR is mainly present in the cytoplasm.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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However, previous reports show that it is also localized to the nucleus.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Within the nucleus, CYTOR functions as an oncogene and is upregulated in multiple human malignancies .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CYTOR also acts as an “endogenous sponge” for several micro-RNAs by binding to them, inhibiting their activity, and promoting malignancy.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Interestingly, CYTOR reportedly regulates cellular actin dynamics and cytoskeletal reorganization in fibroblasts by controlling the expression of genes of the actin polymerization machinery .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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However, the functional importance of CYTOR in CD4 T cells and in the context of HIV infection has not been studied.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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We next conducted gain and loss-of-function studies in J-Lat 6.3 T cells to determine the role of CYTOR in regulating HIV gene expression.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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To achieve CYTOR over-expression, cells were transduced with a lentivirus that drives the expression of CYTOR—exons 1, 4, and 5, the most abundantly expressed form in humans .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Following antibiotic selection, resistant J-Lat 6.3 T stable cells were subjected to RT-qPCR and exhibited a significant increase in CYTOR RNA levels relative to control cells (Fig 2A; blue bar versus grey bar).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Reducing CYTOR expression (knockdown; KD) was also achieved by transducing J-Lat 6.3 T cells with a lentivirus encoding a CYTOR-targeting small-hairpin RNA (shRNA), resulting in a significant decrease of CYTOR RNA levels relative to control cells, expressing a scrambled shRNA as measured by RT-qPCR (Fig 2A; red bar versus grey bar).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Parallel FACS-based analysis of GFP expression in HIV-infected J-Lat 6.3 cells, as a measure of viral gene transcription, revealed that in the absence of T-cell stimulation, no effects on HIV gene expression were observed upon modulation of CYTOR expression.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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However, following T cell stimulation with P/I, CYTOR over-expression led to a relatively small 2-3-fold increase of HIV GFP expression over control cells (Fig 2B; compare blue to grey bars).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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In contrast, depletion of CYTOR led to a 5-fold decrease in HIV gene expression over control cells (Fig 2B; compare red to grey bars).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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HIV GFP expression in control cells expressing scrambled shRNA was unaffected (Fig 2B; grey bar). (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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A).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Modulation of CYTOR RNA levels in J-Lat 6.3 cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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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).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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RNA levels were normalized to gapdh and presented relative to control cells expressing scrambled shRNA (grey bar).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Statistical significance is based on calculating ±SD of data points from four independent experiments using two-way ANOVA. ***
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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p≤0.05. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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B) Effects of CYTOR on HIV gene expression.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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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).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Statistical significance is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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p≤0.05. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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C) Kinetics of latency establishment in the context of CYTOR expression.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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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.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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GFP expression was then followed over time as a measurement of entry into latency.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Statistical significance is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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p≤0.05 and ns: not significant.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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We also followed the establishment of HIV latency post-T cell activation, documenting HIV-GFP expression in control 2D10 cells that expressed scramble shRNA or in cells where CYTOR expression levels were modulated.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Like J-Lat 6.3 cells, 2D10 cells serve as a Jurkat-based latency cell model that carries a minimal Tat-Rev cassette in the context of a GFP reporter under the regulation of the HIV promoter.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Upon T-cell stimulation of control 2D10 Jurkat cells, we confirmed that the expression of HIV-GFP was significantly induced.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Control and CYTOR-modulated stimulated 2D10 Jurkat cells were sorted based on their HIV-GFP expression, obtaining a relatively pure cell population with 100% HIV-GFP expression levels.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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We then monitored latency establishment by following GFP expression in the context of control or CYTOR-modulated cells (Fig 2C).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Our FACS analysis revealed that lower CYTOR levels were associated with a rapid establishment of latency relative to control cells (Fig 2C; grey versus red lines).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Conversely, CYTOR-over-expression enhanced latency reversal, as determined by the elevated levels of HIV-GFP expression that remained relatively high for an extended period following T cell stimulation (Fig 2C; blue line).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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These results suggest that CYTOR expression activates HIV gene expression, significantly reversing latency in 2D10 cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Next, we shifted our analysis to CD4+ primary T cells isolated from healthy donors and the natural target cells for HIV infection.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Depletion of CYTOR in primary human CD4+ T cells was achieved by first stimulating purified cells with anti CD3/CD28 beads and IL2 and then transducing them with a lentivirus encoding a CYTOR-specific shRNA.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Lentivirus driving the expression of scrambled shRNA was used as a control (Fig 3A; n = 3).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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RT qPCR confirmed a significant decrease in CYTOR expression RNA levels relative to control cells that expressed the scramble shRNA (Fig 3B).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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The next day, CYTOR-depleted CD4+ primary stimulated cells (KD) or control cells were transduced with HIVGKO, which codes for a codon-optimized GFP reporter under the control of the HIV-1 promoter and in the context of expression of all viral proteins, and a mKO2 reporter under the control of the constitutive promoter EF10 α .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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HIVGKO transduction can be analyzed by FACS two days later, monitoring parallel transduction efficiency (mKO2+), as well as HIV gene expression (GFP+).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Upon transduction of stimulated primary CD4+ T cells, KD of CYTOR led to decreased HIV gene expression, as monitored by reduced levels of HIV-GFP-expressing cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Conversely, the proportion of cells that expressed EF10 α -mKO2 was slightly elevated in control or CYTOR KD-expressing cells, implying that transduction efficiencies were not affected due to CYTOR depletion but rather specifically drove HIV into a latency state (Fig 3C and 3D). (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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A).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Experimental workflow overview for isolating primary CD4+ T cells.
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