Reactivation of latent HIV-1 provirus via targeting protein phosphatase-1.

Mudit Tyagi,Michael Petukhov,Andrey V Ilatovskiy,Kahli Smith,Aykut Üren,Andrey Ivanov,Tatyana Ammosova,Fatah Kashanchi,Dmytro Kovalskyy,Namita Kumari,Sergey Iordanskiy,Sergei Nekhai,Denitra Breuer,Yasemin Saygideğer Kont

doi:10.1186/s12977-015-0190-4

Abstract

BackgroundHIV-1 escapes antiretroviral drugs by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. This provirus presents the major hurdle in HIV-1 eradication and cure. Transcriptional activation, which is prerequisite for reactivation and the eradication of latent proviruses, is impaired in latently infected T cells due to the lack of host transcription factors, primarily NF-κB and P-TEFb (CDK9/cyclin T1). We and others previously showed that protein phosphatase-1 (PP1) regulates HIV-1 transcription by modulating CDK9 phosphorylation. Recently we have developed a panel of small molecular compounds targeting a non-catalytic site of PP1.ResultsHere we generated a new class of sulfonamide-containing compounds that activated HIV-1 in acute and latently infected cells. Among the tested molecules, a small molecule activator of PP1 (SMAPP1) induced both HIV-1 replication and reactivation of latent HIV-1 in chronically infected cultured and primary cells. In vitro, SMAPP1 interacted with PP1 and increased PP1 activity toward a recombinant substrate. Treatment with SMAPP1 increased phosphorylation of CDK9’s Ser90 and Thr186 residues, but not Ser175. Proteomic analysis showed upregulation of P-TEFb and PP1 related proteins, including PP1 regulatory subunit Sds22 in SMAPP1-treated T cells. Docking analysis identified a PP1 binding site for SMAPP1 located within the C-terminal binding pocket of PP1.ConclusionWe identified a novel class of PP1-targeting compounds that reactivate latent HIV-1 provirus by targeting PP1, increasing CDK9 phosphorylation and enhancing HIV transcription. This compound represents a novel candidate for anti-HIV-1 therapeutics aiming at eradication of latent HIV-1 reservoirs.

Highlights

human immunodeficiency virus (HIV)-1 escapes antiretroviral drugs by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus
Activation of HIV‐1 by protein phosphatase‐1 (PP1)‐targeting compounds We recently developed a small molecule, 1E7-03, that targets a non-catalytic site of PP1, interfered with the binding of Tat to PP1 and inhibited HIV-1 transcription and replication [31]
2-(benzenesulfonyl)-3,4-dihydro-1H-isoquinoline matched aromatic option of the 1H4 (Figure 1a). Such compounds and their analogs available from Enamine stock were grouped in a library containing 38 new compounds which were screened by utilizing a single round HIV-1 infection assay using CEM T cells infected with VSVG-pseudotyped HIV-1 virus expressing luciferase (HIV-1 Luc) as previously described [32]

Summary

Introduction

HIV-1 escapes antiretroviral drugs by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. We and others previously showed that protein phosphatase-1 (PP1) regulates HIV-1 transcription by modulating CDK9 phosphorylation. Efficient HIV-1 transcription from HIV-1 long terminal repeat (LTR) requires both host cell factors and HIV-1 Tat protein [2]. Tat facilitates the formation of super-elongation complex (SEC) at HIV-1 LTR, which, in addition to P-TEFb, carries additional elongation factors and co-activators [18, 19]. Enzymatic activity of P-TEFb and its interaction with Tat is regulated by phosphorylation of CDK serine/threonine residues located in the regulatory T-loop [11]. A recent study by Jonathan Karn and colleagues showed that phosphorylation of CDK9 Ser175 occurs during the induction of latent HIV-1 provirus and that Tat Lys forms a hydrogen bond with CDK9’s phosphoSer175 [24]. Sequestration of PP1 through the expression of nuclear inhibitor of PP1 reduced HIV-1 transcription [29]

Methods

Results

Conclusion