introduction the persistence of replication - competent, but transcriptionally inhibited hiv - 1 proviral dna in long - lived, latent cellular reservoirs is a significant barrier to the development of a functional cure [ 1, 2 ] even after long - term, suppressive antiretroviral therapy ( art ), spontaneous reactivation of proviral gene expression from the latent reservoir is sufficient to initiate viral rebound shortly after art cessation, thus requiring life - long adherence [ 3 – 5 ]. multifaceted and heterogenous blocks to viral gene expression establish and maintain hiv - 1 latency at the epigenetic, transcriptional, and post - transcriptional levels [ 2 ]. several strategies to either reactivate latent proviruses and clear infected cells ( i. e., “ shock and kill ” ) or reinforce latency to prevent spontaneous reactivation ( i. e., “ block and lock ” ) are currently under investigation [ 6, 7 ]. while many small molecule latency reversing agents ( lras ) have been described that reactivate latent proviruses ex vivo, they have had little success in clinical trials [ 8, 9 ]. this failure is partly due to the latent reservoir ’ s heterogenous nature, such that treatment by a single agent that acts to target a specific block may only ever reactivate a small fraction of proviruses in vivo [ 10 ]. even if transcriptional reactivation is achieved, it is unlikely this is sufficient to result in the clearance of these infected cells without additional immune augmentation such as the administration of antibod ies, vaccines, or immunotherap ies to prime the immune system [ 6 ]. given these limitations, much research is now focused on combinatorial approaches to trigger more widespread and robust reactivation [ 11 ]. for example, a recent study reported synergistic reactivation potential between an activator of non - canonical nf - kb signaling ( azd5582 ) and bet bromodomain inhibitors that act to lift blocks to transcriptional initiation and elongation, respectively [ 12 ]. blocks to transcriptional elongation are major contributors to establishing and maintaining hiv - 1 latency [ 13, 14 ]. after integration of the proviral dna, rna polymerase ii ( rna pol ii ) is recruited to the transcription start site by transcription factors that bind cis - elements in the hiv - 1 promoter region. after transcriptional initiation, rna pol ii synthesizes 20 – 60 nucleot ides before stalling through a well - conserved process known as promoter - proximal pausing [ 15 ]. pausing is enforced by several negative elongation factors, including negative elongation factor ( nelf ), drb sensitivity inducing factor ( dsif ), and the rna polymerase ii associated factor 1 ( paf1 ) complex [ 16 – 18 ]. pause release is regulated by positive transcription elongation factor - b ( p - tefb ), a heterodimeric protein complex compose d of cyclin - dependent kinase 9 ( cdk9 ) and cyclin t1 ( ccnt1 ) [ 19 – 21 ]. p - tefb phosphorylates the c - terminal tail of rna pol ii and several negative elongation factors, which collectively license transcriptional elongation [ 22 – 24 ]. recruitment of p - tefb to sites of nascent transcription is a highly regulated process mediated by several cellular complexes. the majority of cellular p - tefb is sequestered in an inactive state by the 7sk ribonucleoprotein ( rnp ) complex [ 25, 26 ]. diverse extracellular stimuli and intracellular signals can induce the release of p - tefb from the 7sk complex [ 27 – 29 ] where it can be recruited to sites of nascent transcription by transcription factors ( i. e., nf - kb and c - myc ) [ 30 – 32 ], epigenetic regulators ( i. e., brd4 ) [ 33, 34 ], or super elongation complexes ( secs ) compose d of an arf4 / fmr2 ( aff ) family scaffold protein in complex with af9, enl, an eleven - nineteen lys - rich leukemia ( ell ) family protein, and an ell - associated factor ( eaf ) protein [ 35 ]. to circumvent this regulatory step, hiv - 1 encodes a trans - activator protein ( tat ) that binds to and recruits p - tefb specifically to sites of nascent proviral transcription through recognition of a transactivation response ( tar ) rna stem loop produced at the immediate 3 ’ end of all viral rna transcripts [ 36 – 38 ]. the distribution of and competition for p - tefb binding among different complexes is an area of active investigation, with several strategies to enhance the biogenesis or availability of p - tefb showing promise for hiv - 1 latency reversal. for example, bet bromodomain inhibitors ( such as jq1 ) have been shown to be potent lras in ex vivo models by releasing p - tefb from brd4 [ 12 ]. likewise, the release of p - tefb from the 7sk rnp complex has been shown to reactivate latent proviruses in ex vivo models [ 39 ]. that being said, the release of p - tefb from the 7sk rnp complex has been shown to directly correlate with increased brd4 binding, suggesting that release from any one complex will not necessarily increase the amount of unbound p - tefb or the amount recruited to specific sites of transcription [ 33 ]. furthermore, post - translational modification of p - tefb ( i. e., through phosphorylation of cdk9 at serine 175 ) has been shown to influence p - tefb distribution in certain regulatory complexes, again highlighting the unique properties of release from each complex [ 40 ]. while the release of p - tefb from the 7sk rnp complex and bet proteins such as brd4 have been explored as strategies for hiv - 1 latency reversal, the release of p - tefb from secs has not been explored. previous work has demonstrated that hiv - 1 tat biochemically co - purifies with several sec proteins [ 41, 42 ], though the reason for this is unclear as they seemingly have functionally redundant purposes in p - tefb recruitment. in this study, we test the hypothesis that the sec is not necessary for hiv - 1 viral transcription and that the release of p - tefb from cellular sec complexes can serve as a novel strategy to reactivate latent hiv - 1 proviruses. results discussion in this study, we demonstrate a potential new strategy for enhancing hiv - 1 latency reversal through the release of p - tefb from the cellular pool of secs. we show that kl - 2, a small molecule inhibitor of the interaction between the sec and p - tefb, is sufficient to enhance viral transcription in primary cd4 + t cells and can synergistically enhance the activity of other lras in certain cell line models of latency. finally, we demonstrate that kl - 2 can increase hiv - 1 gag expression in pbmcs from plwh on suppressive art, most notably in combination with the non - canonical nf - kb agonist, azd5582. we propose a model in which kl - 2 release of p - tefb from the cellular pool of secs enhances transcriptional elongation of integrated proviruses, akin to bet bromodomain inhibitors and 7sk rnp inhibitors ( fig 6d ). these results have several implications for our understanding of viral transcription and future directions. latency reversal through the release of p - tefb from cellular secs had not been previously explored, likely due to the perceived dependency of viral transcription on the sec. this effect has been supported by biochemical purifications of hiv - 1 tat from human cell lines that revealed interactions with a larger sec [ 41, 42 ], as well as by genetic knock - down experiments in cell lines showing a decrease in tat - dependent transcription upon sec component depletion [ 64, 65 ]. however, given that both the sec and tat recruit p - tefb to sites of nascent transcription, they share some functional redundancy. we found that knock - out of sec components from activated, primary cd4 + t cells from 12 independent donors did not inhibit viral replication, suggesting that the sec is not required in this cellular context. this result was independently verified using kl - 2, which inhibits the interaction between ccnt1 ( p - tefb ) and aff1 / 4 ( of the larger sec ).