Structural and functional study of HIV-1 Tat interaction with 7SK RNP

About This Grant

Project Summary Despite decades of research effort, the HIV-1 retrovirus remains a major global public health crisis. To date, there is no cure for HIV-1, with an infected individual requiring continuous antiretroviral therapy (ART) to thwart the onset of AIDS. When ART is ceased, there is a rebound of viremia that originates from the pool of latently infected CD4+ T cells. This rebound within a latently infected cell begins with transcriptional reactivation of the HIV-1 genome, and the Transactivator of transcription (Tat) protein lies at the heart of HIV-1 gene expression. Tat activates gene expression by recruiting the kinase Positive Transcription Elongation Factor b (P-TEFb) to a paused RNA Polymerase II (RNAP II), ultimately promoting RNAP II pause release. Specifically, Tat recruits P- TEFb to the nascent Transactivation Response (TAR) RNA element, with Tat positioning P-TEFb for phosphorylation of the RNAP II C-terminal domain and transcription factors to promote RNAP II pause escape. Importantly, a major cellular reservoir of P-TEFb is held inactive by the dimeric Hexim1 (or less commonly Hexim2) protein within the 7SK ribonucleoprotein (RNP), and prior work supports that Tat “hijacks” P-TEFb from 7SK RNP for subsequent P-TEFb recruitment to TAR. However, the molecular mechanisms underpinning this hijacking remain uncharacterized, posing a fundamental barrier to targeting Tat transactivation as an HIV-1 eradication strategy. A potential route to Tat removal of P-TEFb is by competing with Hexim1 for 7SK RNA binding, since one of the Hexim1 binding sites on 7SK RNA mimics the Tat recognition site on TAR RNA. Therefore, Aim 1 proposes to define the competition between Tat and Hexim1 for binding to 7SK RNA in vitro by NMR, ITC, and EMSA experiments, building upon recent work from the Feigon lab that successfully employed these same methods to identify Hexim1 binding sites on and affinities for 7SK RNA. Aim 2 proposes to purify and biochemically characterize a Tat-bound 7SK RNP from mammalian cells, since this complex is a proposed intermediate prior to Tat–P-TEFb handover to TAR. Specifically, the complex will be characterized with mass photometry, mass spectrometry, and SHAPE-/DMS-MaP. Lastly, Aim 3 describes how the structure of a Tat– 7SK RNP will be determined by cryoEM and how structure-derived hypotheses will be tested on Tat transactivation in a cell line model of HIV-1 latency. The proposed work is supported by the Feigon lab’s excellent track record of combining NMR and cryoEM to study challenging, dynamic RNPs and, all the necessary facilities and equipment are available to execute this work. Together, the goal of this fellowship proposal is to uncover the structural and mechanistic basis of Tat “hijacking” of P-TEFb from the 7SK RNP, providing new insights to aid design of HIV-1 therapeutics and offering a rich training in structural biology methods.