Background: Strategies to achieve a cure for HIV/AIDS will require the therapeutic control of HIV latency whether they involve “shock and kill” activation or “deep latency” repression of HIV transcription. We have previously reported that the HIV core promoter is selectively recognized by cellular pre-initiation complexes of HIV (PICH) via its TATA box and adjacent sequences of HIV essential for Tat trans-activation (TASHET) element. To uncover molecular targets to therapeutically control latency we have now identified protein components of PICH and characterized their impact on HIV transcription.
Methods: DNA affinity chromatography coupled to mass spectrometry analysis was used to identify host cell PICH proteins that recognize TASHET. Co-immunoprecipitation was used to determine interacting partners of identified PICH proteins. Biophysical techniques including fluorescence anisotropy and circular dichroism (CD) were used to demonstrate direct PICH-DNA interactions. Chromatin immunoprecipitation (ChIP) assays were used to test PICH interactions with the HIV promoter in cellulo. siRNA were employed to test the impact of PICH proteins on HIV transcription in cellulo. HIV reporter viruses were used to test the requirement of PICH-binding sequences of TASHET for reactivation by latency-reversing agents (LRA) in infected T cells.
Results: We identified two new proteins in nuclear extracts from uninfected PBMC, HeLa cells, and Jurkat T cells that specifically bind to the HIV core promoter in vitro. These proteins, termed PICH-130 and PICH-37, interact tightly in cells as demonstrated by co-immunoprecipitation. PICH-130 and PICH-37 form part of a HDAC complex of previously unknown function that contains HDAC1 and HDAC2. Strikingly, PICH-37 directly and selectively binds the TATA and adjacent CTGC motifs within TASHET in vitro. PICH-37 binds the HIV promoter in infected HeLa and Jurkat cells. siRNA-mediated knock-down of PICH-130 and PICH-37 strongly reduced both Tat-activated and basal HIV transcription in cellulo. Intriguingly, mutation of the PICH-37-binding CTGC motifs in reporter virus completely blocked reactivation of HIV by pharmacological LRA that act via the Tat/TAR/7SK axis in T cells, but only partially reduced reactivation by HDAC inhibitors.
Conclusions: PICH-37 and PICH-130 form part of a novel HDAC complex that controls HIV latency and is essential for reactivation via the Tat/TAR/7SK pathway.