identification of differentially expressed genes and clustering analysisdifferentially expressed genes between es and eb were identified by anova at p value 0. 05 using bioconductor [ 17 ]. unsupervised hierarchical clustering analysis and principal component analysis ( pca ) were conducted using software pcluster [ 31 ] and treeview [ 18 ]. identification of diagnost ic markerspam ( prediction analysis of microarray ) was employed for the identification of diagnost ic markers from insulin pathway genes by using the software package bioconductor [ 17 ]. pam is a class prediction method for expression data mining. it can provide a list of significant genes whose expression characterizes each diagnost ic class. the average gene expression level in multiple classes, such as es, eb, ns, and fb, was divided by the within - class standard deviation for that gene. the nearest centroid classification computed by pam takes the protein expression profile from a new sample, and compares it to each of these class centroids [ 26 ]. rt - pcr and quantitative real - time pcr analysistotal rna was isolated with trizol ( invitrogen. cdna was synthesized using 2. 5 μg total rna in a 20 - μl reaction with superscript ii ( invitrogen ) and oligo ( dt ) 12 – 18 ( promega; madison, wi ). one microliter rnase h ( invitrogen ) was added to each tube and incubated for 20 minutes at 37°c before proceeding to the rt - pcr analysis. the pcr primers are: rps4y - forward: 5 ' agattctcttccgtcgcag 3 ', rps4y - reverse, 5 ' ctccaccaatcaccatacac 3 '; eifay - forward, 5 ' ctgctgcatcttagttcagtc 3 '; eifay - reverse 5 ' cttccaatcgtccatttccc 3 '. quantitative real time pcr gene specific primer pairs and probes were purchased from applied biosystems ( foster city, ca ) for the following genes: mmp3 ( hs00233962 _ m1 ), tfrsf11b ( hs00171068 _ m1 ), thbs1 ( hs00170236 _ m1 ), krtha4 ( hs00606019 _ gh ), and for internal control β - actin ( actb, hs99999903 _ m1 ). introduction the introduction of antiretroviral therapy ( art ) has successfully limited the spread of human immunode ficiency virus ( hiv ) and improved patient clinical outcomes. however, a complete cure for hiv infection remains out of reach, as the transcriptionally silent but replication - competent provirus that is integrated into the host genome persists in long - lived cellular reservoirs, which are comprised of memory - resting cd4 + t cells, as well as cells of myeloid lineages [ 1, 2 ]. these reservoirs are highly stable and are resistant to both art and the effects of the host immune surveillance, thus posing a significant obstacle to eradicating the hiv reservoirs. consequently, in most people living with hiv, interrupting art leads to rapid viral load rebound, usually within weeks after treatment cessation [ 3 – 6 ]. as t cell stimulation triggers activation of proviral transcription, one strategy that has been proposed to eliminate the hiv reservoirs is a “ shock - and - kill ” approach, which utilizes latency - reversing agents ( lras ) to first activate dormant hiv - infected t cells and facilitate cell death by viral cytopathic effects or immune-mediated killing. this step is done in the presence of art, so there are no further rounds of hiv replication. [ 7 – 9 ]. alternatively, a “ block and lock ” approach frees infected individuals from art by silencing hiv transcription and inducing a deep state of latency. nevertheless, despite promising therapeutic options, these strategies and others have regretfully failed to achieve significant clinical efficacy. 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 ]. epigenetic constraints that suppress proviral gene transcription are essential for establishing hiv latency [ 16, 17 ]. 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 [ 18, 19 ]. within the infected t cells, gene transcription of the integrated provirus and the host genome are synchronized [ 20, 21 ]. both display key steps of gene transcription, which include initiation, promoter arrest, and elongation. 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 ]. 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 [ 27 ]. long non - coding rnas ( lncrnas ) are transcripts with longer than 200 nucleot ides that lack protein - coding capacity. to date, over 200, 000 cell type - specific lncrnas have been identified and display critical regulatory functions of many processes within cells [ 28 – 31 ]. however, the functions of most of these transcripts remain poorly understood. in the context of hiv, roles for several cellular lncrnas have been documented [ 32 – 40 ]. moreover, significant gaps still remain in our knowledge about the mechanistic roles that lncrnas play in cd4 t cell activation and hiv latency. 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 ). 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. cytor directly binds the hiv promoter and activates viral gene transcription and latency reversal by recruiting p - tefb to the viral promoter. cytor also exerts its effects indirectly by controlling global gene expression along with actin dynamic pathways, thereby affecting t cell activation and hiv infection. results discussion 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 - ). our analysis show that different transcriptional profiles exist in cells where hiv is activated versus cells where it remains latent. cytor lncrna was identified as one of these rnas, and its expression is elevated upon t cell stimulation, where hiv is active. these observations were further confirmed in primary cd4 + t cells ( fig 1 ). functional analyses show that following t cell stimulation, over - expression of cytor activates hiv gene expression, while its depletion inhibits viral 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 ). effects of cytor on hiv infection and latency establishment were also confirmed in stimulated primary cd4 + t cells ( fig 3 ). 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. 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 [ 56 ].