Targeting Tat Inhibitors in the Assembly of Human Immunodeficiency Virus Type 1 Transcription Complexes

Abstract

Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein, which relieves a block to elongation by recruiting an elongation factor, P-TEFb, to the viral promoter. Here, we report the discovery of potent Tat inhibitors that utilize a localization signal to target a dominant negative protein to its site of action. Fusing the Tat activation domain to some splicing factors, particularly to the Arg-Ser (RS) domain of U2AF65, creates Tat inhibitors that localize to subnuclear speckles, sites where pre-mRNA processing factors are stored for assembly into transcription complexes. A U2AF65 fusion named T-RS interacts with the nonphosphorylated C-terminal domain of RNA polymerase II (RNAP II) via its RS domain and is loaded into RNAP II holoenzyme complexes. T-RS is recruited efficiently to the HIV-1 promoter in a TARindependent manner before RNAP II hyperphosphorylation but not to cellular promoters. The "preloading" of T-RS into HIV-1 preinitiation complexes prevents the entry of active Tat molecules, leaving the complexes in an elongation-incompetent state and effectively suppressing HIV-1 replication. The ability to deliver inhibitors to transcription complexes through the use of targeting/localization signals may provide new avenues for designing viral and transcription inhibitors.

Autophagy Restricts HIV-1 Infection by Selectively Degrading Tat in CD4 + T Lymphocytes

“…Since Tat is absolutely required for viral transcription, many therapeutic strategies targeting this viral protein have been considered, such as Tat antagonists (52), dominant negative Tat (53,54), Tat-inhibitory peptides (55)(56)(57)(58), or siRNAs against Tat (59)(60)(61). However, none of these strategies has been successful to date.…”

Transition Step during Assembly of HIV Tat:P-TEFb Transcription Complexes and Transfer to TAR RNA

“…Chromatin immunoprecipitation (ChIP) assays were performed as previously described (21) using a HeLa cell line containing the HIV:RRE promoter driving FFL expression transfected with CycT1N-Rev in the absence or presence of Tat AD using calcium phosphate. The cells were incubated for 36 h and washed in PBS before cross-linking and lysis (23).…”

“…1D and data not shown). Tat is known to activate transcription when tethered to the nascent transcript through another protein (Rev), using a heterologous protein-RNA interaction (Tat-Rev-RRE) (23,102). In this context, the Tat-Rev fusion activates transcription to a level comparable to CycT1N-Rev and T-RS, thus highlighting the importance of the Tat AD, but not the RBD, during Tat activation of RNA-bound P-TEFb (Fig.…”

“…1B), an RBD-deficient Tat protein (T-RS) excludes wild-type Tat from the promoter by preferentially assembling with P-TEFb through the Tat AD but cannot facilitate transfer of P-TEFb to TAR, thus blocking transition to elongation (23). Therefore, this reporter assay monitors Tat:P-TEFb preassembly at the promoter irrespective of TAR and Tat RNA-binding activity.…”

“…Comparison between inhibition of Tat activation and the standard Tat activation assay (Fig. 1A) allows the uncoupling of Tat:P-TEFb requirements for promoter assembly versus transition to the nascent RNA and thus may define a step prior to TAR binding (21,23,43,68).…”