An activator of G protein-coupled receptor and MEK1/2-ERK1/2 signaling inhibits HIV-1 replication by altering viral RNA processing
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.