The mTOR Complex Controls HIV Latency

Abstract

A population of CD4 T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and in vitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat and via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.

Keywords: HIV LTR; HIV latency; HIV transcription; genome-wide shRNA screen; latency reversal; mTOR inhibition; reactivation from latency.

Figure 1. High Complexity shRNA Screen to Identify Genes that Control HIV Latency

(A) Schematic of strategy used to stimulate J-Lat 5A8 cells with CD3/CD28 to promote HIV exit from latency (B) Strategy to introduce human genome-wide mCherry-tagged shRNA library into J-Lat cells, and then stimulate cells with a 3 µg/ml CD3/CD28 to yield 15% double-positive cells. GFP and mCherry represent J-Lat 5A8 HIV-GFP, and shRNA expression, respectively. (C) Calculations conducted on samples that are deep sequenced to obtain p-values to identify genes involved in HIV latency. (D) FANCC, an example of a gene that promotes latency. (E) CAPN10, an example of a gene that inhibits latency. (F, G) Graphs depicting number of enriched (F) or disenriched (G) genes plotted as a function of signed log 10 P values. See also Tables S1, S2 and S3

Figure 2. CORUM Analysis Results in the Identification of Several Interesting Complexes

(A) Schematic of procedure used to calculate and identify latency-promoting complexes and latency-inhibiting complexes in CORUM using p-values. (B) (Top) Latency-promoting and (Bottom) latency-inhibiting complexes (see text for details). See also Table S4

Figure 3. CRISPRi against MLST8 in latent K562 cells prevents reversal of HIV latency by LRAs

(A) Procedure to obtain latent CRISPRi K562 cells and transduce them with sgRNA lentiviruses and select by puromycin. LRAs were added to test reactivation of HIV. (B) Efficiency of MLST8 knockdown with three different sgRNAs checked by western blot. Cells transduced with NC (negative control) sgRNA lentiviruses done in duplicate (NC-1 and NC-2) were used as control. (C, D) Percentage of GFP-positive cells 24 h after reactivation with PMA (C) and Ingenol-B (D). Data are represented as mean +/− SD of triplicate values, representative of two independent experiments. (E) Simple scheme representing the mTORC1 and mTORC2 subunits and regulator that were knockdown by CRISPR interference. (F,G,H,I) Latent CRISPRi K562 cells were transduced with sgRNA lentiviruses targeting MTOR (F), RICTOR (G), RAPTOR (H), TSC1 (I) and selected by puromycin. Percentages of GFP-positive cells 21–24 h after reactivation with PMA are indicated on the upper panels. Efficiency of knockdown for each gene is shown on the lower panels with western blot. Cells transduced with NC (negative control) sgRNA lentivirus were used as control. Data are represented as mean +/− SEM of at least three independent experiments. See also Figure S1

Figure 4. mTOR Pathway Inhibitors Suppress Reactivation of Latent HIV in Human CD4 T Cells

(A) Percentage of GFP-positive K562 cells expressing NC or MLST8 sgRNAs 21–24 h after PMA stimulation with or without simultaneous Torin1 treatment. Data are represented as mean +/− SD of four independent experiments. (B) Detection of selected phosphorylated proteins in CD4 T cells from four independent donors using PathScan analysis. CD4 T cells were treated with either 0.01% DMSO or incubated for 30 minutes with 25µL of αCD3/αCD28 activating beads with DMSO, 250nM pp242, 97.7nM Torin1 or 10nM rapamycin. The amount of phosphorylated proteins is scaled internally to each donor. (C–E) Bcl-2 transduced latently infected cells were either unstimulated (yellow) or stimulated with CD3/CD28 antibodies (2.5 µg/ml CD3 and 0.65 µg/ml CD28 (orange); 10 µg/ml CD3 and 0.65µg/ml CD28 (red)) for 48 h to reactivate HIV in the presence of increasing concentrations of pp242 (C), Torin1 (D) and rapamycin (E). Reactivation of HIV was assessed by measuring GFP by flow cytometry, and the percentages of reactivation were calculated for each batch of latently infected cells by maximum activation with PMA and Ionomycin (top panels) (Yang et al., 2009). Percentage of live cells in each sample is shown (bottom panels). Data are represented as mean +/− SD. See also Figure S2

Figure 5. mTOR Inhibition Suppresses Tat-Independent and Tat-Dependent HIV LTR Activation and CDK9 Phosphorylation

(A–C) pp242 (A), Torin1 (B) and rapamycin (C) suppress Tat-dependent HIV LTR activation after 24h in a dose-dependent manner in luciferase assays with the HIV LTR construct in Jurkat cells. Tat was used at increasing doses (0, 0.05, 0.25 and 1.25 ng). Data are represented as mean +/− SD of triplicate values of relative luciferase units (RLU) normalized by protein content (representative of at least two independent experiments). (D) pp242, Torin1 and rapamycin treatment differentially affects Tat-dependent HIV LTR activation in the context of integrated provirus (TZM-bl cells). Tat was used at increasing doses (0, 1, 10 and 100 ng plasmid). TZM-bl cells were transfected with indicated amounts of Tat plasmid and treated with DMSO, Torin 1 (100, 200nM), pp242 (0.5, 1µM), or Rapamycin (10, 50nM). 24h post-treatment, cells were lysed and HIV transcription was measured via luciferase activity. Data are represented as mean +/− SEM of fold change of RLU normalized by protein content. (E) pp242, Torin1 and Rapamycin reduce basal LTR activity in A72 Jurkat cells (integrated LTR-GFP) measured by percentage of GFP-positive cells. (F–H) Primary CD4 T cells were isolated from a donor’s blood and either pre-treated with pp242 (0, 8, 40, 200 and 1000 nM) for 30 minutes (F), left untreated (G), or pre-treated with Okadaic acid (0, 10, 100 or 1000 nM) for 30 minutes (H). The cells were co-stimulated with CD3/CD28 antibodies for 2 h. Cells were harvested for protein extraction right before (0 min) and after (120 minutes) adding the CD3/CD28 beads. Proteins were extracted as explained in Experimental Procedures. Whole cell protein extracts from (G) were either phosphatase-treated or not. Extracts were run on an SDS-PAGE gel and immunoblotted for the indicated proteins (CDK9, phospho-S6 (Ser240/244) and S6). See also Figure S3

Figure 6. mTOR Inhibition Causes Suppression of Reactivation of Latently Infected Cells in the Patient Model

(A) pp242- and Torin1-treated CD4 T cells in three HIV-infected patients on anti-retroviral therapy with undetectable viral load led to a drastic decrease in fold change of HIV mRNA. (B) Patient samples in (A) were regraphed to show the percentage reactivation of HIV mRNA. Significant p-values with t-test (two-sample unequal variances) are shown (**: p<0.01)