The Long Noncoding RNA<i>HEAL</i>Regulates HIV-1 Replication through Epigenetic Regulation of the HIV-1 Promoter

Abstract

A major challenge in finding a cure for HIV-1/AIDS is the difficulty in identifying and eradicating persistent reservoirs of replication-competent provirus. Long noncoding RNAs (lncRNAs, >200 nucleotides) are increasingly recognized to play important roles in pathophysiology. Here, we report the first genome-wide expression analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs). We identified an lncRNA, which we named <u>H</u>IV-1-<u>e</u>nh<u>a</u>nced <u>l</u>ncRNA (<i>HEAL</i>), that is upregulated by HIV-1 infection of MDMs, microglia, and T lymphocytes. Peripheral blood mononuclear cells of HIV-1-infected individuals show elevated levels of <i>HEAL</i> Importantly, <i>HEAL</i> is a broad enhancer of multiple HIV-1 strains because depletion of <i>HEAL</i> inhibited X4, R5, and dual-tropic HIV replications and the inhibition was rescued by <i>HEAL</i> overexpression. <i>HEAL</i> forms a complex with the RNA-binding protein FUS, which facilitates HIV replication through at least two mechanisms: (i) <i>HEAL</i>-FUS complex binds the HIV promoter and enhances recruitment of the histone acetyltransferase p300, which positively regulates HIV transcription by increasing histone H3K27 acetylation and P-TEFb enrichment on the HIV promoter, and (ii) <i>HEAL</i>-FUS complex is enriched at the promoter of the cyclin-dependent kinase 2 gene, <i>CDK2</i>, to enhance CDK2 expression. Notably, <i>HEAL</i> knockdown and knockout mediated by RNA interference (RNAi) and CRISPR-Cas9, respectively, prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment <i>in vitro</i> Our results suggest that silencing of <i>HEAL</i> or perturbation of the <i>HEAL</i>-FUS ribonucleoprotein complex could provide a new epigenetic silencing strategy to eradicate viral reservoirs and effect a cure for HIV-1/AIDS.<b>IMPORTANCE</b> Despite our increased understanding of the functions of lncRNAs, their potential to develop HIV/AIDS cure strategies remains unexplored. A genome-wide analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs) was performed, and 1,145 differentially expressed lncRNAs were identified. An lncRNA named <u>H</u>IV-1-<u>e</u>nh<u>a</u>nced <u>l</u>ncRNA (<i>HEAL</i>) is upregulated by HIV-1 infection and promotes HIV replication in T cells and macrophages. <i>HEAL</i> forms a complex with the RNA-binding protein FUS to enhance transcriptional coactivator p300 recruitment to the HIV promoter. Furthermore, <i>HEAL</i> knockdown and knockout prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment, suggesting <i>HEAL</i> as a potential therapeutic target to cure HIV-1/AIDS.