HIV-1 Tat Forms a Dissociable Complex with 7SK snRNP and Exhibits Clade Differences in Reversing the Sequestration of...

Recent efforts to employ ectopic Tat as a latency-reversal strategy can benefit from the identification of Tat variants with more efficient transactivation activity. Tat primarily serves as a recruiter of the host transcription elongation factor P-TEFb to proviral HIV to stimulate transcription elongation. Tat can also functionally interact with a host of chromatin-modifying enzymes to promote HIV transcription initiation. In actively dividing T cells, P-TEFb is kept inactive by the 7SK snRNP complex. Although Tat is capable of physically extracting P-TEFb from 7SK snRNP as well as forming an assembly with the sequestrator complex, it is unclear whether Tat:7SK snRNP (Tat:7SK) is a reversible functional intermediate that mediates the recruitment of P-TEFb to proviral HIV. This study explored the reversibility of Tat:7SK and whether Tat clade variants differ in their ability to assemble this complex, mobilize P-TEFb and transactivate latent HIV. While the ectopic expression of clade B NL4-3 Tat caused a substantial dissociation of P-TEFb from 7SK snRNP, affinity tag-based immunoprecipitation experiments confirmed the assembly of Tat:7SK with the displacement of the P-TEFb inhibitory component HEXIM1. The P-TEFb kinase inhibitor flavopiridol (FVP), known to rapidly dissociate P-TEFb from 7SK snRNP, also induced the efficient release of Tat:P-TEFb from Tat:7SK, suggesting that P-TEFb remains a component of this latter complex. While Tat variants representing clades A, B, C and CRF_AE exhibited comparable binding to P-TEFb, clade D Tat showed the least binding. Unlike clades B and CRF_AE, clades A and C Tat associated much more weakly with the core 7SK snRNP subunits MEPCE and LARP7, indicating that their bound P-TEFb is likely liberated. Evaluation of latent HIV reactivation in the Jurkat 2D10 clonal model revealed a rapid loss of Tat transactivation capacity over time across all clades examined, with the most striking disparity observed between Clades A and D. Overall, our findings reveal substantial clade-specific differences in Tat's ability to liberate P-TEFb from 7SK snRNP and transactivate latent HIV, with the clade A variant displaying the highest potency. Contrary to the prevailing paradigm that Tat:7SK is a "dead-end" complex that is unable to avail P-TEFb for transcriptional control, we show that it is a reversible intermediate from which Tat:P-TEFb can be released en bloc.