Fig 1.
5342191 inhibits HIV-1 gene expression and replication.
(A) 5342191 chemical structure. HeLa rtTA-HIV-ΔMls (B and D-E) or CD4+ 24ST1NLESG T cells (C) were treated with indicated concentrations or IC90 (2 μM) of 5342191 (191), or DMSO (control) only for 4 h prior to Dox or PMA (+) induction (resp.) of HIV-1 expression for 20 h. Each treatment contained equal concentrations of DMSO solvent. After ~24 h, cell supernatants were harvested for (B-C) p24CA ELISA of HIV-1 Gag expression (black diamonds) and XTT assay of cell viability (gray circles; n ≥ 4–5, mean, s.e.m.) while (D-E) cell lysates (~30 μg) were analyzed by immunoblot for expression of (D) HIV-1 structural proteins: Gag (p55, p41, and p24) and Env (gp160 and gp120), (E) viral regulatory factors: Rev (p19) and Tat (p16 and p14), and internal loading controls: GAPDH or α-tubulin (n ≥ 4–6, mean, s.e.m.). Position of molecular weight (MW) standards were marked adjacent to each gel/blot. Lanes in (D-E, resp.) were cropped/assembled from the same blots (S2A and S2B Fig). (F-G) Primary CD4+ T cells (PBMCs) were infected or left uninfected (yellow circles) with HIV-1 Ba-L, treated with 3 μM 5342191 (191, green diamonds), 3.7 μM AZT (red boxes), or DMSO only (black circles), which was partially replenished with fresh drug & media after 4 days, and cell supernatant harvested every 2 days for p24CA ELISA to monitor effects on (F) HIV-1 growth over 0–6 days and (G) dose-response of 0–3 μM of 5342191 on HIV-1 replication (red circles) and cell viability (gray boxes, by trypan blue exclusion) on day 6. Data from (F-G) are n = 4 from 1 donor, representative of 4 different donors, mean, s.e.m. (H-I) CEM-GXR cells were infected with WT (IIIB or N54) or RT inhibitor (i), PRi, INi, or CRi-resistant strains of HIV-1 described in (H), treated with 0, 0.15, 0.3, 0.6, 1.25, 2.5, or 5 μM of 5342191 (gradient bar, except CRi: 0–1.25 μM), and their effects on (I) HIV-1 LTR activation (red circles) and cell viability (gray boxes) quantified from GFP fluorescence and live-cell counts, respectively, by flow cytometry after 3 days of culture (n ≥ 3, except CRi: n ≥ 2–3, mean, s.e.m.). Dotted-black lines in (G and I) mark 100% cell viability or IC50. All results are relative and statistically compared to treatment with 0 μM of compound.
Fig 2.
5342191 affects HIV-1 RNA processing and expression/modification of SR splicing factors with limited perturbation of AS and expression of host RNAs.
HeLa rtTA-HIV-ΔMls (A-B and E-H), 24ST1NLESG T (C), and HeLa rtTA-HIV(Gag-GFP) cells (D) and (I-K) were treated with/without IC80-IC90 of 5342191 (2, 4, 2, and 2.5 μM, resp.) and Dox per Fig 1 and assayed as follows. (A-C) Quantitation of the relative expression of HIV-1 US (gray), SS (white), and MS RNAs (black) in cells by qRT-PCR. (A) Diagram of the HIV-1 genome indicating position of primers used in amplification. Solid arrow heads denote start while dashed-lined arrows represent exon coverage. (B-C) Graph of RNA levels quantified from HeLa rtTA-HIV-ΔMls and 24ST1NLESG T cells (resp.) treated with/without 5342191 (n ≥ 3, mean, s.e.m.). Results are relative and statistically compared to DMSO (+) for each RNA class. (D) Trafficking of US RNAs (labeled with Texas Red) detected by FISH (representative of n ≥ 3). Nuclei were detected by DAPI stain, Gag-GFP expression by GFP, and images captured at 630x magnification. (E-H) RNA-Seq quantifying the AS of host RNAs (mean PSI) from 9,806 exon inclusion/exclusion events examined and DE genes [mean fold change (Δ)] from 11,406 host RNAs detected from 5342191 or DMSO-treated cells (S1 and S3 Tables; n = 2, mean). (E) Scatterplot of PSIs displaying differences in AS between 5342191 (y-axis) and DMSO (x-axis) with significant ΔPSIs (p <0.05) indicated by colored circles as follows: <10% (gray), 10–20% (yellow), and ≥ 20% (red). (F) Total number and percentage (%) of AS events altered (ΔPSI ≥ 10% and 20%), (G) total number and % of DE genes changed (≥ 2, 5, and 10 fold), and (H) Venn diagram of the AS (≥ 10%) and DE genes (≥ 2 fold) affected in common. (I, J, and S5 Fig) Representative immunoblots and (K) graph quantifying the accumulation (and modification) of endogenous SR proteins from lysates of treated cells (~30 μg, n ≥ 3, mean, s.e.m.). Results are relative and statistically compared to DMSO (+). β-actin (B-C) and Stain-Free-labeled total proteins (I-K) served as internal loading controls for normalization of RNA and protein data, respectively.
Fig 3.
5342191 reduction of Tat expression is reversed by inhibition of the proteasome and has limited effects on the abundance of host proteins.
HeLa rtTA-HIV-ΔMls cells were treated with/without 2 μM 5342191 and Dox per Fig 1 and viral/host protein levels quantified as follows. (A-B) Cells treated with/without 5342191 and Dox for 24 h and with/without addition of proteasome-inhibitor MG132 8 h prior to harvest were immunoblotted for HIV-1 CA (p24 Gag, white), p16 (purple) and p14 Tat (blue), and gp160 (pink) and gp120 Env (red) from lysates (~30 μg). (A) Representative immunoblots and (B) multi-bar graph of these results (n ≥ 3, mean, s.e.m.). Results in (A-B) are relative to DMSO (+) and cells treated with 5342191 and MG132 were statistically compared to treatments with this compound and no MG132 as illustrated by a black-dashed line for one of the targets. (C-E) Averaged normalized ion intensities (log2) from 5,326 proteins identified by TMT LC-MS/MS (S4 Table) were analyzed as follows. (C) Proteins from 5342191 (y-axis, n = 3) and DMSO (+)-treated cells (x-axis, n = 2) were plotted to illustrate differences in abundance between treatments with the levels of each change depicted by colored circles as follows: <1.5-fold Δ (+/- 0–0.59, gray), 1.5-2-fold Δ (+/- 0.59–0.99, orange), and >2-fold Δ (+/- 1.0+, green). Degree of correlation (r, Pearson) and a blue-dashed line representing a drug with no impact on protein abundance are shown. (D) Total number and percentage (%) of proteins affected by 5342191 that were ≥ 1.5 and 2-fold changed from control. (E) Venn diagram comparing proteins with ≥ 1.5-fold Δ between 5342191, 9147791, and DMSO (-) from data in (D) and S9C and S9D Fig, respectively.
Fig 4.
Inhibition of HIV-1 gene expression by 5342191 is reversed by blocking MEK1/2-ERK1/2 or GPCR signaling.
HeLa rtTA-HIV(Gag-GFP) cells were pretreated with/without a kinase inhibitor prior to treatment with 2 μM 5342191 or DMSO and Dox per Fig 1 to determine the signaling pathway(s) involved by monitoring kinase activation and/or recovery of Gag-GFP expression in cell lysates. Cells were pretreated with/without a kinase inhibitor (and confirmed to block each kinase) as follows: MEKi #1 (12 μM U0126), MEKi #2 (5 μM Selumetinib), JNKi (1.25 μM SP600125), or p38i (15 μM SB203580) overnight for ~15 h (A and/or alongside previous study [28]); [Ca2+]i (5 μM BAPTA-AM) or NCXi (5 μM KB-R7943) for 3 h (B); and Gαi (~10 μM BIM-46187,), EGFRi (120 nM PD158780,), Srci (350 nM Herbimycin A), or PI3Ki (10 μM LY294002) for 3 h (Fig 5A on Ras activity and/or S12E Fig via EGF effects on HIV-1 expression). Impact of each of these inhibitor combinations on cell density were monitored in S12B, S12D, and S12F Fig. Tubulin or Stain-Free-labeled total proteins served as internal loading controls and for normalization of these data. (A) Graph quantitating the activation levels of ERK1/2 (with/without MEKi #1 or #2), JNK1/2/3, MK-2, and p38 in treated cells (n ≥ 3–5, mean, s.e.m.) as observed and described in representative blots provided in S11A–S11C Fig. (B) Graph quantifying relative [Ca2+]i from fluorescence of Fura Red AM loaded into cells treated with/without 5342191 (n ≥ 4, mean, s.e.m.). Ouabain treatment served as a positive control in this assay [28]. (C-D) Graphs quantitating rescue of Gag-GFP expression in treated cells by detecting fluorescence from reducing SDS-PAGE (n ≥ 3–4, mean, s.e.m., and initially from live/fixed-plated cells of S12A and S12C Fig) as described in representative gels of S11C–S11F Fig. Note that data on the right side of (C) contains a second experiment set for MEKi #2. (E) Graph quantifying the level of HIV-1 RNAs in treated cells by qRT-PCR (n ≥ 3–4, mean, s.e.m.). Assay was performed as described in Fig 2A–2C. All results from (A-E) are relative to DMSO (+) and no kinase inhibitor (as well as for each RNA class for qRT-PCRs). Statistical comparisons were performed as illustrated by gray/black-dashed lines (and asterisks) for one of the targets. A horizontal gray-dashed line marks the level of US and SS RNAs in 5342191-treated cells with no kinase inhibitor.
Fig 5.
Exogenous expression of variants of the small G protein, N-Ras, inhibits HIV-1 gene expression.
(A-D) HeLa rtTA-HIV(Gag-GFP) cells were (A) treated with/without 5342191 per Fig 1 or (B-D) transfected with WT, oncogenic (12D), or dominant-negative (17N) N-Ras, or mock plasmid and co-transfected with (+) tTA (or without, -) to activate HIV-1 expression. After 48 h, cell lysates were analyzed as follows. (A) Graph of the relative Ras activity (relative RLUs) in lysates (45 μg) of cells treated with/without 5342191 quantified by Ras-GTP ELISA (n ≥ 3, mean, s.e.m.). (B) Representative gel of Gag-GFP expression detected by GFP fluorescence from reducing SDS-PAGE and representative immunoblots of ERK1/2 (phospho and total), HA-tagged N-Ras, and α-tubulin/GAPDH assayed from 30 μg of cell lysates (representative of n ≥ 3–5). (C-D) Graphs quantifying the level of expression of (C) HIV-1 Gag described in (B, n ≥ 3–5, mean, s.e.m.) and (D) HIV-1 RNAs detected by qRT-PCR as described in Fig 2A and 2B (n ≥ 3, mean, s.e.m.). (E-F) Models of the antiviral signal (E) and impact (F) of 5342191 (191) on HIV-1 gene expression in cells. This study describes a new small molecule inhibitor (5342191) which, in a similar manner as CSs, activates (arrows) MEK1/2-ERK1/2 signaling to inhibit (block arrows) HIV-1 replication (E-F). Unlike CSs which function via the Na+/K+-ATPase, 5342191 activates Gα proteins of GPCRs at the cell membrane (E). Although 5342191 also stimulates JNK and MK-2 (a target of p38 MAPK), it achieves suppression of HIV-1 gene expression without initiating p38 MAPK or NCX-mediated [Ca2+]i flux observed with CSs (E, detailed in S15 Fig). 5342191 treatment of cells decreases accumulation of HIV-1 incompletely-spliced (US/SS) RNAs (and increases MS RNAs, progressively-red arrow; F) through activation of GPCR and MEK1/2-ERK1/2 signals described in (E) and impedes buildup of essential viral regulatory proteins (Tat and Rev, F). This results in blocked expression and cytoplasmic transport (loss of Rev) of US and SS HIV-1 RNAs which encode key HIV-1 structural (Gag and Env) and regulatory proteins (p14 Tat) that are essential for HIV-1 replication (F). Addition of this compound also reduces accumulation of HIV-1 regulatory factors in cells by altering translation of MS RNAs and/or degradation of viral regulatory factors such as Tat (F). This new inhibitor (5342191) achieves potent suppression of HIV-1 replication with little/no changes in cell viability, alternative splicing and expression of host RNAs, or nascent synthesis and abundance of host proteins.