notably, disrup tion of the sec using kl - 2 resulted in significant increases in hiv - 1 replication in primary cd4 + t cells whereas genetic knockout of most sec members had minimal to no impact on replication. one potential explanation for this difference is that genetic knockout results in the ablation of sec assembly and relocalization of p - tefb into other complexes at steady-state whereas chemical perturbation by kl - 2 results in the release of p - tefb from secs that are continually being formed. understanding the dynamics of p - tefb distribution and relocalization upon different types of perturbation is an ongoing area of investigation. while we expected kl - 2 to enhance the transcriptional elongation of integrated proviruses due to the release of p - tefb from cellular secs, we also saw increases in transcriptional initiation as measured by qrt - pcr for tar transcript levels. likewise, in j - lat 5a8 cells, we saw increases in transcriptional elongation, transcriptional initiation, and in rna pol ii recruitment to the proviral promoter when kl - 2 was added, even though kl - 2 addition alone was not sufficient for reactivation as measured by gfp positivity in this model. this finding suggests that either kl - 2 has secondary effects not mediated by p - tefb or that the redistribution of p - tefb away from secs has secondary effects that could impact transcriptional initiation at proviral integration sites. bet bromodomain inhibitors have also been reported to increase hiv - 1 transcriptional initiation [ 12, 13 ], though these effects have been suggested to occur through modulation of the epigenetic regulatory functions of these proteins [ 66 ]. other reports have indicated that p - tefb mediated release of paused rna pol ii can result in enhanced transcriptional initiation and even rna pol ii recruitment simply by increasing the number of transcribing polymerases [ 67 ]. still, this connection between transcriptional elongation and initiation has yet to be fully understood in the context of hiv - 1 transcription. while kl - 2 was sufficient to boost viral replication in activated, primary cd4 + t cells, in and of itself it displayed minimal reactivation potential in both cell line models of latency and in pbmcs from plwh on suppressive art. this finding is similar to our recent report of a novel inhibitor of the paf1 complex ( ipaf1c ) that had minimal activity on its own, but greatly enhanced the reactivation potential of other lras [ 16 ]. both cases highlight the multifaceted nature of the blocks to viral gene expression that underlie the latent state as well as the limitations to single agent drug screening to identify promising, next - generation lras. combinatorial approaches to dissect the genetic underpinnings of hiv - 1 latency and discover new, synergistic drug interactions should be prioritized. while kl - 2 alone failed to significantly increase hiv - 1 gag transcript levels in patient pbmcs, in combination with azd5582 it resulted in a 16 - fold increase over azd5582 treatment alone and a 53 - fold increase over the dmso control. crosswise dose titrations in j - lat 5a8 cells showed a strong synergistic potential between azd5582 and kl - 2. this is consistent with reports of robust synergy between azd5582 and p - tefb release through bet bromodomain inhibition [ 12 ]. notably, the bet bromodomain inhibitor jq1 showed minimal reactivation activity in our patient pbmcs, even in the presence of kl - 2, in contrast to our cell line data. this finding could reflect stochastic differences driven by variations in patient characteristics, integration site, chromatin state, transcription factor availability, etc. [ 13 ]. future work will compare p - tefb release from secs to release from other cellular reservoirs, such as brd4 or the 7sk rnp. recent studies have shown that post - translational modifications of p - tefb, most notably phosphorylation of cdk9 serine 175 and threonine 186, can drive inclusion into different complexes and may strongly influence bioavailability and activity [ 40, 68 – 70 ]. therefore, it is possible that disrup tion of complexes housing ‘ active ’ p - tefb is a more direct route to redirecting p - tefb activity. this is not to say that the sec is never required for viral transcription. in latency model cell lines that lacked a functional tat ( u1 cells ) or tar stem loop ( ach - 2 cells ), kl - 2 inhibited the reactivation potential of several lras, suggesting that viral transcription may be more dependent on the sec when tat is either defective or not expressed. we attempted to test this by inhibiting tat in the j - lat 5a8 model cell line using two previously described tat - dependent transcription inhibitors, triptolide and spironolactone. while both compounds reduced lra efficacy, kl - 2 still boosted the activity of jq1 and pma, but not azd5582. this suggests that p - tefb can be recruited to proviral integration sites in a tat and sec - independent manner upon pma or jq1 treatment, potentially through a transcription factor such as nf - kb. the inability of azd5582 to do so suggests that non - canonical nf - kb activation does not recruit the same milieu of transcription factors, making it uniquely tat or sec dependent. this is consistent with the complete lack of activity of azd5582 in cell line models lacking functional tat / tar activity and may underlie the remarkable synergy between non - canonical nf - kb agonists and agents that release p - tefb [ 12 ]. additionally, triptolide has been characterized outside of hiv - 1 transcription in its ability to prevent rna pol ii reinitiation following pausing through inhibition of xeoderma pigmentosum group b - complementing protein ( xpb ) and is often used as a tool compound for measuring the fate of paused rna pol ii at different time points [ 51, 71 ]. with this in mind, it is possible that compounds jq1 and pma result in de novo recruitment of rna pol ii thereby increasing transcriptional initiation whereas azd5582 may be more reliant on rna pol ii pause - release. to further explore the tat dependency of kl - 2, we tried to rescue tat function in the u1 cell line using a dox - inducible system. we hypothesized that by providing tat, the sec would no longer be required for viral gene expression such that sec disrup tion by kl - 2 would enhance reactivation as seen in the j - lat models. while tat induction itself was sufficient for reactivation, this reactivation was completely abolished by the addition of kl - 2. even when tat was minimally induced and other lras were added, kl - 2 still inhibited reactivation, suggesting that additional factors — such as steady-state levels of p - tefb, integration site, epigenetic factors, or transcription factor availability — may drive sec dependency besides just the presence or absence of tat. indeed, sec disrup tion by kl - 2 in the original report of the inhibitor demonstrated an outsized impact on myc - dependent transcription [ 32 ], suggesting that additional factors driving the sec dependency of proviral transcription have yet to be described. regardless, the dual - acting nature of kl - 2 in enhancing latency reactivation in some circumstances ( i. e., if tat is present ) and inhibiting latency reactivation in others ( i. e., when the cell state dictates sec dependency ) presents a unique opportunity to leverage the heterogenous nature of the latent reservoir to both reverse and promote latency. taken together, our results indicate that release of p - tefb from cellular secs is a novel mechanism for promoting hiv - 1 viral transcription during both active and latent infection. we demonstrated the enhancement of latency reversal in multiple latent cell line models and in primary pbmcs from plwh on suppressive art.