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

Abstract

The ability of HIV-1 to evolve resistance to combined antiretroviral therapies (cARTs) has stimulated research into alternative means of controlling this infection. We assayed >60 modulators of RNA alternative splicing (AS) to identify new inhibitors of HIV-1 RNA processing-a segment of the viral lifecycle not targeted by current drugs-and discovered compound N-[4-chloro-3-(trifluoromethyl)phenyl]-7-nitro-2,1,3-benzoxadiazol-4-amine (5342191) as a potent inhibitor of both wild-type (Ba-L, NL4-3, LAI, IIIB, and N54) and drugresistant strains of HIV-1 (IC 50 :~700 nM) with no significant effect on cell viability at doses tested. 5342191 blocks expression of four essential HIV-1 structural and regulatory proteins (Gag, Env, Tat, and Rev) without affecting total protein synthesis of the cell. This response is associated with altered unspliced (US) and singly-spliced (SS) HIV-1 RNA accumulation (~60% reduction) and transport to the cytoplasm (loss of Rev) whereas parallel analysis of cellular RNAs revealed less than a 0.7% of host alternative splicing (AS) events (0.25-0.67% by � 10-20%), gene expression (0.01-0.46% by � 2-5 fold), and protein abundance (0.02-0.34% by � 1.5-2 fold) being affected. Decreased expression of Tat, but not Gag/ Env, upon 5342191 treatment was reversed by a proteasome inhibitor, suggesting that this compound alters the synthesis/degradation of this key viral factor. Consistent with an affect on HIV-1 RNA processing, 5342191 treatment of cells altered the abundance and phosphorylation of serine/arginine-rich splicing factor (SRSF) 1, 3, and 4. Despite the activation of several intracellular signaling pathways by 5342191 (Ras, MEK1/2-ERK1/2, and JNK1/2/3), inhibition of HIV-1 gene expression by this compound could be reversed by pre-treatment with either a G-protein α-subunit inhibitor or two different MEK1/2 inhibitors. These observations demonstrate enhanced sensitivity of HIV-1 gene expression to small changes in host

Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency

“…Although TG003 had no effect, chlorhexidine effectively suppressed HIV-1 gene expression in micromolar doses in our cell-based assays, suggesting distinct roles of CLKs in modulating viral gene expression [36]. Multiple small molecule modulators of RNA processing, digoxin [37], chlorhexidine [36], 8-azaguanine [40], 5350150 [40], ABX464 [41], 1C8 [35], 9147791 [42], and 5342191 [43], are able to alter HIV-1 RNA accumulation and inhibit virus replication with very limited alterations to host RNA processing [35,[41][42][43][44]. Together, these observations highlight the delicate balance in viral RNA processing needed for HIV-1 because they affect distinct steps of transcription/RNA processing.…”

“…Several studies have focused on modulating cellular splicing factors such as SR and hnRNP proteins to alter HIV-1 gene expression [23,81,82]. The identification of multiple small molecules (digoxin, ABX464, didehydro-cortistatin A, 8-azaguanine, 5310150, 1C8, 791, 191, filgotinib, GPS491) that inhibit HIV-1 gene expression post-integration by modulating viral RNA processing with limited impact on host cell viability demonstrates the feasibility of this approach [35,37,[40][41][42][43][83][84][85].…”

A New Approach to 3D Modeling of Inhomogeneous Populations of Viral Regulatory RNA

“…Tertiary modeling of polypeptide conformations has been integral to elucidating the functional mechanisms of enzymes, chaperones, many structural proteins and receptor-ligand interactions and for in silico modeling of small molecule therapeutics. Tertiary modeling of RNA structures has robust potential to guide the rational design of antiviral therapeutics [ 58 , 59 , 60 , 61 ]. Supportive evidence that alternative nt–nt pairings propagate structural changes throughout the HIV-1 leader RNA includes TAR-binding compounds exert structural effects outside TAR [ 62 , 63 ] and small molecule binding within the CES reduce virus titer [ 64 ].…”

The Thiazole-5-Carboxamide GPS491 Inhibits HIV-1, Adenovirus, and Coronavirus Replication by Altering RNA Processing/Accumulation

“…With GPS491 addition increasing the levels of Tat mRNA, the basis for the loss of Tat p16 protein (encoded by Tat1 mRNA) was not immediately clear. An alternative explanation for the loss of Tat protein expression is that GPS491 induced changes in the rate of Tat synthesis and/or degradation as seen previously with other modulators of HIV-1 replication [19,23]. To test this hypothesis, we examined whether treatment with the proteasome inhibitor MG132 could restore Tat protein expression.…”