this work demonstrates the importance of increasing the production or availability of free p - tefb for recruitment to viral loci as a powerful strategy for bolstering current lras, most notably non - canonical nf - kb agonists. due to the heterogeneity of blocks to viral replication in the latent reservoir, it is likely that combinatorial lra treatments will be the best strategy for potent latency reversal moving forward. further efforts are needed to understand the intracellular distribution of active p - tefb to characterize the most critical reservoir to target to enhance transcription. additionally, our work demonstrates that disrup tion of secs could enhance latency reversal or promote the maintenance of latency depending on the cellular context. understanding the mechanism that controls this molecular switch would aid in understanding whether sec disrup tors could be a viable dual - acting molecule to aid in finding a functional cure for hiv - 1 infection. methods flow cytometry and analysis of viability / infection / reactivation data flow cytometry analysis was performed on an attune nxt acoustic focusing cytometer ( thermo fisher scientific ), recording all events in a 40 - μl sample volume after one 150 μl of mixing cycle. data were exported as fcs3. 0 files using attune nxt software v3. 2. 0 and analyzed with a consistent template on flowjo. briefly, cells were gated for lymphocytes by light scatter followed by doublet discriminat ion in both side and forward scatter. cells with equal fluorescence in the bl - 1 ( gfp ) channel and the vl - 2 ( amcyan ) channel were identified as autofluorescent and excluded from the analysis. a consistent gate was then used to quantify the fraction of remaining cells that expressed the target of interest. analysis of hiv - specific rna transcripts following cdna synthesis, viral transcripts were assessed by qrt - pcr as described previously [ 13 ]. for hiv - 1 tar, the following primers were used: pf: 5 ′ - gtctctctggttagaccag - 3 ′; pr: 5 ′ - tgggttccctagytagcc - 3 ′; and probe: 5 ′ - agcctgggagctc - 3 ′. hiv - 1 long ltr levels were assessed using the following primers: pf: 5 ′ - gcctcaataaagcttgccttga - 3 ′; pr: 5 ′ - gggcgccactgctagaga - 3 ′; and probe: 5 ′ - ccagagtcacacaacagacgggcaca - 3. for expression level normalization, β - actin was used ( thermo fisher scientific, catalog no. 4331182 ). notably, β - actin is an rna pol ii controlled gene. to ensure that β - actin levels were not being altered by kl - 2 induced sec disrup tion, we compared β - actin expression to that of the 18s ribosomal subunit, an rna pol i controlled gene ( thermo fisher scientific, catalog no. 4331182 ). no statistically significant changes were noted between the two housekeeping genes upon kl - 2 treatment, so β - actin was used as the normalization gene for subsequent qpcr analyses. the reaction was performed using taqman fast advanced master mix ( thermo fisher scientific, catalog no. 4444553 ) according to manufacturer ’ s instructions. briefly, 10 μl of reaction was mixed using 5 μl of taqman master mix, 0. 5 μl of 20x primer probe mix ( 18 μm of primers and 5 μm probe ), 2. 5 μl water, and 2 μl of template cdna. the pcr cycles were as follows: 50 °c for 2 minutes, 95 °c for 20 seconds, followed by 40 cycles of 95 °c for 1 second and 60 °c for 20 seconds. rna sequencing analysis sequencing data was demultiplexed and trimmed using trimmomatic v0. 36 to remove adapters and low - quality reads. trimmed reads were aligned to the homo sapiens reference genome grch38 and transcripts quantified using the hisat2 - stringtie pipeline [ 76 ]. differential gene expression analysis of the quantified gene transcripts was performed with deseq2 v. 1. 42. 0 r package using r v. 4. 3. 2. after retaining genes with nonzero total read count and with more than 10 reads in total between all samples, we fitted a model that included all treatments to account for overall variability and identified differentially expressed genes ( degs ) within that model between all tested conditions against dmso - treated cells ( i. e. kl2 vs dmso, azd5582 vs dmso, and azd5582 + kl2 vs dmso ). to define degs, we used as cut - offs an absolute log2 fold change > 1 and a false discovery rate ( fdr ) comparecluster function. for these analyses all genes whose gene symbols could be mapped to entrez ids using the org. hs. eg. db v. 3. 18. 0 bioconductor annotation package were included. statistical analysis all statistical analysis was performed using graphpad prism version 10. 2. 0 ( 392 ) for windows 64 - bit, graphpad software, boston, massachusetts, usa ( www. graphpad. com ). results dcas9 - targeted hiv - 1 ltr - interactome analysis identified prmt3 as a ltr - binding factor seeking host factors that specifically associate with the hiv - 1 ltr, we conducted an ltr - interacted proteome analysis based on a refined version of a previously described nuclease - deficient cas9 ( dcas9 ) - targeted chromatin - based purification strategy ( clasp ) ( cas9 locus - associated proteome ) ( fig. 1a ) 33, 34. the screen used nh1 cells, which is a modified hela cell line that harbors an integrated hiv - 1 ltr - driven luciferase reporter gene in the host genome35 ( fig. 1b ). to get the most effective sgrnas that target ltr, we used two methods for screening. we individually inserted the sgrnas into the cas9 plasmid, which was then co - transfected with a tat - expression plasmid into nh1 cells that harbors the ltr - driven luciferase reporter gene. if a sgrna - cas9 combination can target and disrup t the ltr sequence, a reduction in luciferase activity is observed. we designed seven sgrnas targeting different regions of the hiv - 1 ltr ( fig. 1b ). an sgrna targeting the yeast gal4 gene was used as the negative control. among the designed sgltrs, the top three sgltrs ( # 5, 6, 7 in pink, fig. 1b ) exhibiting the capacity to attenuate the ltr driven - luciferase expression activated by hiv - 1 tat ( fig. 1c ). then, we performed anti - flag chip - qpcr in cells transfected with the sgltrs - dcas9 - 3 × flag plasmids to further verify the abilities of the sgltrs to target dcas9 - 3 × flag to the ltr, and found that sgltr - 1 and - 2 failed to recruit dcas9 to ltr efficiently ( supplementary fig. 1a, b ), sgltr - 5 and - 6 displayed the most significant effect, whereas sgltr - 3, - 4 and - 7 all displayed a similar and partial effect ( supplementary fig. 1c, d ). thus, sgltr - 5, - 6, - 7 were selected for subsequent chip - qpcr analysis. by utilizing the combination of the three sgrnas, we found that in comparison to sggal4, these sgrnas can guide dcas9 to the ltr as revealed by chip - qpcr analysis ( fig. 1d ). next, we performed in vitro transcription reactions to generate these sgrnas, which were then used for dcas9 / sgrnas complex formation. this complex was subsequently incubated with sheared chromatin that contained the ltr - bound proteins for dcas9 - 3 × flag immunoprecipitation, following with mass spectrometry analysis. fig. 1 * * * dcas9 - targeted hiv - 1 ltr - interactome analysis identified prmt3 as a ltr - binding factor. * * * a schematic of dcas9 - targeted proteome analysis workflow. ( 1 ) sgrnas targeting the ltr were complex formation with dcas9 - 3 × flag; ( 2 ) crosslinking of proteins bound to the ltr; ( 3 ) dcas9 - 3 × flag - sgrna complex was incubated with sheared fragments from nh1 cells that stably expressed ltr - luciferase reporter; ( 4 ) immunoprecipitation was used to capture ltr bound proteins; ( 5 ) ltr - interacted proteins were identified by mass spectrometry. b schematic of the position of 7 sgrnas. c luciferase activity was measured in nh1 cells transfected with a plasmid encoding each sgrna and control sgrna ( sggal4 ), which expresses the cas9 in the presence of tat ( p = 0. 0016, p = 0. 0024, p = 0. 0042 ).