Inhibition of the H3K27 demethylase UTX enhances the epigenetic silencing of HIV proviruses and induces HIV-1 DNA hypermethylation but fails to permanently block HIV reactivation.

Kien Nguyen,Jonathan Karn,Won-Kyung Cho,Benjamin Luttge,Curtis Dobrowolski,Meenakshi Shukla,Bryan R Cullen

doi:10.1371/journal.ppat.1010014

Abstract

One strategy for a functional cure of HIV-1 is "block and lock", which seeks to permanently suppress the rebound of quiescent HIV-1 by epigenetic silencing. For the bivalent promoter in the HIV LTR, both histone 3 lysine 27 tri-methylation (H3K27me3) and DNA methylation are associated with viral suppression, while H3K4 tri-methylation (H3K4me3) is correlated with viral expression. However, H3K27me3 is readily reversed upon activation of T-cells through the T-cell receptor. In an attempt to suppress latent HIV-1 in a stable fashion, we knocked down the expression or inhibited the activity of UTX/KDM6A, the major H3K27 demethylase, and investigated its impact on latent HIV-1 reactivation in T cells. Inhibition of UTX dramatically enhanced H3K27me3 levels at the HIV LTR and was associated with increased DNA methylation. In latently infected cells from patients, GSK-J4, which is a potent dual inhibitor of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A, effectively suppressed the reactivation of latent HIV-1 and also induced DNA methylation at specific sites in the 5'LTR of latent HIV-1 by the enhanced recruitment of DNMT3A to HIV-1. Nonetheless, suppression of HIV-1 through epigenetic silencing required the continued treatment with GSK-J4 and was rapidly reversed after removal of the drug. DNA methylation was also rapidly lost after removal of drug, suggesting active and rapid DNA-demethylation of the HIV LTR. Thus, induction of epigenetic silencing by histone and DNA methylation appears to be insufficient to permanently silence HIV-1 proviral transcription.

Highlights

Despite effective long-term suppression of viral loads by combination antiretroviral therapy, latent HIV-1 may quickly rebound upon cessation of treatment [1, 2]
UTX/KDM6A is a specific H3K27 demethylase that selectively targets H3K27me3 and H3K27me2
Since H3K27me3 formation by EZH2 is a powerful repressive mechanism to silence HIV-1 [22, 23], we reasoned that knockdown of UTX could be used to enhance HIV-1 transcriptional silencing and perhaps establish a permanently silenced provirus

Summary

Introduction

Despite effective long-term suppression of viral loads by combination antiretroviral therapy (cART), latent HIV-1 may quickly rebound upon cessation of treatment [1, 2]. The first, often referred to as “kick and kill” [3], is based on the concept that latent HIV1, once pharmacologically reactivated by latency reversing agents (LRAs), could subsequently be eliminated by cytotoxicity and/or the enhanced immunological surveillance [4, 5]. An alternative approach, referred to as “block and lock”, is designed to permanently suppress the rebound of quiescent HIV-1 by inducing epigenetic silencing [14,15,16]. In the best documented example of a “block and lock” strategy, the Valente laboratory has reported that didehydro-cortistatin A (dCA), an inhibitor of Tat-mediated HIV-1 transcription, can enhance epigenetic silencing of the HIV-1 promoter, leading to long-term inactivation of the provirus [14, 17,18,19]. Regardless of the chosen strategy, a more comprehensive understanding of the mechanism involved in HIV-1 latency is urgently required when designing effective HIV-1 cure strategies

Methods

Results

Conclusion