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