d chip - qpcr analyses were conducted to assess the occupancy of 3 × flag - dcas9 at the promoter ( left ) or nascent region ( right ) of ltr when cells were transfected with control plasmid, a mixture of plasmids containing sgltrs - 5, 6, 7 or the sggal4 plasmid ( promoter: p = 0. 0009, p = 0. 0011, nascent: p = 0. 000004, p = 0. 000003 ). e proteins showing over 1. 5 - fold enrichment relative to the control sample identified from 3 × flag - dcas9 immunoprecipitation following by mass spectrometry analysis were listed. f chip - qpcr analyses were conducted to assess the occupancy of prmt3 at ltr - luciferase region ( p = 0. 0001, p = 0. 0012, p = 0. 0005, p = 0. 0008 ). the schematic display of the four primers was shown on the top of the panel. g chip - qpcr analyses were conducted to assess the occupancy of prmt3 at ltr - luciferase regions in the absence or presence of tat ( p = 0. 0022, p = 0. 0012, p = 0. 0045, p = 0. 0017 ). h cut & tag sequencing was performed in primary cd4 + t cells isolated from virologically suppressed hiv - 1 - infected patient cells that were treated with pma, by using igg or prmt3 antibod y. the alignment results of sequencing with the hiv - 1 genome in three patients were displayed. error bars = mean + / − sd of three biological replicates. source data are provided as a source data file. mass spectrometry detected a total of 28 proteins with at least 1. 5 - fold enrichment relative to the non - targeting negative control ( fig. 1e and supplementary data 1 ). prmt3, which has been shown to function in transcriptional regulation and antiviral innate immunity15, 30, 36, was among the hits with the highest fold enrichment of sgltr / sggal4, and was therefore selected for subsequent investigation. we performed chip - qpcr analysis to examine the binding of prmt3 to the ltr. prmt3 was significantly enriched at ltr - luciferase reporter region ( fig. 1f ), which can be further increased in the presence of tat ( fig. 1g ). the binding of prmt3 at the ltr was also verified in jurkat 2d10 cells ( supplementary fig. 2 ). notably, we performed cleavage under targets & tagmentation ( cut & tag ) sequencing by using igg or prmt3 antibod y in cd4 + t cells isolated from hiv - 1 infected patient cells, and demonstrated that while igg displayed undetectable signal at the ltr, prmt3 showed significant binding signal around ltr ( fig. 1h ). thus, these results demonstrated that prmt3 is an hiv - 1 ltr binding factor, which is further enhanced in the presence of tat. discussion the elusive nature of latent hiv - 1 reservoirs presents a formidable challenge to eradicating the virus from infected individuals49 – 51. despite the various strategies employing either the latency reversal agents ( lras ) or latency promoting agents ( lpas ), which are aimed at either purging or deeply silencing the latent viral reservoirs, these agents have yet to be made into effective drugs for curing hiv / aids and targets are still needed for the development of therapies. furthermore, the premise of using the current and future versions of lras or lpas for treating hiv - 1 hinges on the potential to epigenetically modulate the hiv - 1 promoter activity, aiming for a sustained activation or suppression of viral transcription, respectively52 – 54. the significance of targeting epigenetic control of viral transcription was highlighted recently in a human clinical trial, where the combination of panobinostat, a potent pan - histone deacetylase inhibitor, with interferon - α2a resulted in an enhanced vulnerability of latent hiv - 1 reservoir cells, underscoring the pivotal role of epigenetic manipulation in combating hiv - 1 persistence2. in the current study, by performing the dcas9 - targeted ltr interactome screening, we have identified prmt3 as a positive regulator to promote hiv - 1 latency reversal. mechanistically, prmt3 activates hiv - 1 transcription by interaction with tead4 and the two proteins co - localize at the ltr by using specific tead4 - binding motifs, thereby regulating chromatin accessibility and facilitating p - tefb / tat recruitment. thus, our findings propose a promising epigenetic strategy to combat latent hiv - 1 infection, underscoring prmt3 and its partners as promising therapeutic targets for anti-hiv - 1 drug development. our study demonstrates tead4 as a positive regulator of hiv - 1 transcription, acting alongside prmt3 through a specific interaction with the ltr ’ s ggaat motif. this discovery expands our current understanding of tead4 ’ s regulatory roles in host cells and suggests its potential regulation of hiv - 1 viral expression. the specificity of tead4 ’ s interaction with the motif containing the core ggaat sequence within the hiv - 1 ltr and its synergy with prmt3 in promoting tat - dependent hiv - 1 transcription and a specific subset of host gene transcription is particularly intriguing given tead4 ’ s established roles in cell survival, proliferation, tissue regeneration, stem cell maintenance, embryonic trophoblast and organ development and tumorigenesis9, 11, 13, 14, 55. specifically, we selected three host genes, prdm1, slitrk5, and tgfb2, for further validation of our proposed prmt3 - tead4 regulation model. our data demonstrate that in addition to the hiv - 1 ltr, prmt3 and tead4 indeed associated with these cellular gene promoters, where the binding of tead4 significantly decreases after the depletion of prmt3, suggesting a common regulatory mechanism involving the prmt3 - tead4 complex that exists at the hiv - 1 ltr, as well as the promoters of selected host genes. at the hiv - 1 ltr, the interaction of the prmt3 - tead4 complex with tat and the p - tefb provides further regulation of hiv - 1 transcription. on cellular gene promoters such as the three genes mentioned above, it is conceivable that transcription can also be modulated through controlling the function and / or binding of the prmt3 - tead4 complex to the ggaat motif in response to changes in physiological or pathological conditions. this co - regulation of viral and host gene transcription by prmt3 - tead4 underscores a finely tuned regulatory mechanism that integrates the cellular and viral transcriptional control. furthermore, tead4 has also been demonstrated to interact with p - tefb, which is the core transcriptional component of the super elongation complex ( sec ) essential for tat - activated hiv - 1 transcription. given the fact that tead4 is a dna sequence - specific transcription factor, it remains to be investigated whether it plays a key role in recruiting sec to both host and hiv - 1 gene promoters that contain the tead4 - binding motif. while previous studies have indicated prmt3 ’ s involvement as a key factor in mediating host responses to viral infection17, 30, its specific role in hiv - 1 infection and latency remains largely unexplored. for instance, in zebrafish, prmt3 has been shown to negatively regulate antiviral responses17. similarly, recent studies utilizing the prmt3 inhibitor, sgc707 treatment, or prmt3 knockout mice have revealed prmt3 ’ s facilitation of hsv - 1 infection30. in conjunction with our current discovery demonstrating prmt3 ’ s promotion of hiv - 1 transcription to reverse latency, these findings suggest that prmt3 may serve as a potential target for broad - spectrum antiviral drugs. however, future comprehensive research is imperative to fully comprehend the extent of prmt3 ’ s involvement in mediating viral - host interactions. it is noteworthy that besides prmt3, other prmts have also been implicated in regulating anti-hiv activity. for instance, prmt6 has been shown to inhibit hiv - 1 replication in vitro by directly methylating several hiv - 1 proteins, including tat, rev, and nucleocapsid protein, thereby interfering with their functions56, 57. similarly, prmt2 was recently discovered to suppress hiv - 1 transcription by methylating tat and promoting the phase separation of p - tefb and tat58.