A high-throughput screening assay for silencing established HIV-1 macrophage infection identifies nucleoside analogs that perturb H3K9me3 on proviral genomes

Abstract

HIV-infected macrophages are long-lived cells that represent a barrier to functional cure. Additionally, low-level viral expression by central nervous system (CNS) macrophages contributes to neurocognitive deficits that develop despite antiretroviral therapy (ART). We recently identified H3K9me3 as an atypical epigenetic mark associated with chronic HIV infection in macrophages. Thus, strategies are needed to suppress HIV-1 expression in macrophages, but the unique myeloid environment and the responsible macrophage/CNS-tropic strains require cell/strain-specific approaches. Here, we generated an HIV-1 reporter virus from a CNS-derived strain with intact auxiliary genes expressing destabilized luciferase. We employed this reporter virus in polyclonal infection of primary human monocyte-derived macrophages (MDM) for a high-throughput screen (HTS) to identify compounds that suppress virus expression from established macrophage infection. Screening ~6,000 known drugs and compounds yielded 214 hits. A secondary screen with 10-dose titration identified 24 meeting criteria for HIV-selective activity. Using three replication-competent CNS-derived macrophage-tropic HIV-1 isolates and viral gene expression readout in MDM, we confirmed the effect of three purine analogs, nelarabine, fludarabine, and entecavir, showing the suppression of HIV-1 expression from established macrophage infection. Nelarabine inhibited the formation of H3K9me3 on HIV genomes in macrophages. Thus, this novel HTS assay can identify suppressors of HIV-1 transcription in established macrophage infection, such as nucleoside analogs and HDAC inhibitors, which may be linked to H3K9me3 modification. This screen may be useful to identify new metabolic and epigenetic agents that ameliorate HIV-driven neuroinflammation in people on ART or prevent viral recrudescence from macrophage reservoirs in strategies to achieve ART-free remission. IMPORTANCE Macrophages infected by HIV-1 are a long-lived reservoir and a barrier in current efforts to achieve HIV cure and also contribute to neurocognitive complications in people despite antiretroviral therapy (ART). Silencing HIV expression in these cells would be of great value, but the regulation of HIV-1 in macrophages differs from T cells. We developed a novel high-throughput screen for compounds that can silence established infection of primary macrophages, and identified agents that downregulate virus expression and alter provirus epigenetic profiles. The significance of this assay is the potential to identify new drugs that act in the unique macrophage environment on relevant viral strains, which may contribute to adjunctive treatment for HIV-associated neurocognitive disorders and/or prevent viral rebound in efforts to achieve ART-free remission or cure.

Keywords: HIV; epigenetics; high-throughput screen; macrophage; silencing.

Fig 1

Primary MDM silencing assay. (A) Schematic of brain-derived macrophage-tropic HIV-1 reporter construct YU2/Δenv/nLuc. The destabilized nano-luciferase (nLuc) reporter was placed upstream of a P2A ribosomal skip site and inserted at the 5’ end of the nef gene of HIV-1 YU2. A premature stop was created in env by introduction of a 2-bp insertion at codon 59 of the open reading frame. (B) Overview of primary cell assay. Monocytes were cultured for 6 d to mature into MDM and then infected with the VSVg-pseudotyped reporter virus. They were cultured for 7 d more to allow infection to proceed, then re-plated into 384 well plates. One day later, test compounds were added and maintained in the cultures for 5 d. Cells were then harvested and nLuc for virus expression and CellGlo for cell viability measured in cell lysates of replicate plates. (C) Timing the completion of integration after reporter virus infection. The integrase inhibitor raltegravir (RTG) was added 1 d prior to, 1 d after, or 4 d after MDM were infected with the VSVg-pseudotyped reporter virus. By 4 d after infection, infection was resistant to integration blocking.

Fig 2

HTS of compounds. Waterfall plot of the HTS primary screen of 4,800 compounds from the SelleckChem library. Compounds were added at 10 μM, and duplicate plates were analyzed in parallel for nLuc expression from integrated provirus in MDM, and for CellTiter-Glo as an indication of cell viability. Compounds were ranked by percent inhibition of nanoLuc (Y-axis) and filtered for <25% change in CellTiter-Glo.

Fig 3

Titration screening of compounds. Compounds were tested at 10 concentrations from 0.1 nM to 10 mM. Representative compounds are shown that displayed dose-dependent separation between nLuc and CellGlo inhibition. Out of 214 compounds subject to titration screening, 24 showed dose-dependent separation between virus inhibition and cell toxicity.

Fig 4

Testing of compounds in MDM infection with different HIV-1 strains. Six-day-old MDM were infected with three replication-competent CNS-derived HIV-1 strains. Five days after infection, efavirenz was added to prevent further rounds of infection. Three days later, compounds were added at four concentrations (0.3, 1, 3, and 10 μM) in the continued presence of efavirenz. Cells were harvested 5 d later and subject to qRT-PCR for expression of the viral tat (blue) and nef (red) transcripts, and cellular GUSB (green). Gene expression is shown as percent inhibition relative to untreated control infections, calculated by the ΔCt method. Infections were carried out using duplicate wells of MDM, and data reflect means ± SEM of three independent infectious using different MDM donors. (A) HIV-1 YU-2 derived from the IMC, carrying the VSVg envelope in mixed pseudotypes to boost infectivity; (B) HIV-1 JRFL; and (C) HIV-1 Jago.

Fig 5

Nucleoside analogs nelarabine and fludarabine reduce HIV transcripts. MDM were infected with YU-2/VSVg and efavirenz added 5 d later to prevent new rounds of infection. Four days later, they were treated with or without nelarabine (3 µM) or fludarabine (3 μM) . RNA was isolated 7 d later and assayed by RT-qPCR with primers targeting HIV tat, gag, nef and two different regions of the 5′ nascent transcript (TAR-short and TAR-long). Transcription is shown as fold over cellular GUSB levels, calculated by the ΔΔCt method.

Fig 6

Nelarabine reduces H3K9me3 on the HIV proviral genome and cellular subtelomeres. MDM were infected with YU-2/VSVg. Seven days later, efavirenz was added to block further rounds of reinfection, and 3 d later treated with or without nelarabine (3 µM). Six days later, crosslinking was carried out and nuclei isolated. Cells were assayed by ChIP for association of H3K9me3, total histone H3, or IgG control using primers positioned throughout the HIV genome, or at control loci in the human genome (actin, GAPDH, subtelomere 10q_CTCF, and 10q_TERRA). (A) HIV-1 genome regions targeted by PCR primers; (B) H3K9me3; (C) Total histone H3; and (D) IgG control. Data are presented as percent of input DNA for each primer pair and presented as means ± SEM for n = 3 technical replicates.