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

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 drug-resistant strains of HIV-1 (IC50: similar to 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 (similar to 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 alpha-subunit inhibitor or two different MEK1/2 inhibitors. These observations demonstrate enhanced sensitivity of HIV-1 gene expression to small changes in host RNA processing and highlights the potential of modulating host intracellular signaling as an alternative approach for controlling HIV-1 infection. HIV-1 resistance to current antiretroviral therapies requires new approaches for managing this infection. We assayed over 60 compounds for new inhibitors of HIV-1 RNA processing-an area of the virus lifecycle not targeted by current drugs and primarily under the control of the host cell-and identified compound 5342191 as a potent inhibitor of multiple wild-type and drug-resistant strains of HIV-1. This new inhibitor suppresses production of four essential HIV-1 structural proteins and regulatory factors by reducing expression of most RNAs encoding them and alters synthesis/degradation of at least one of these factors. Inhibition of HIV-1 gene expression by 5342191 resulted from activation of intracellular signaling from G-protein coupled receptors. This study not only exploits a weakness that we previously identified in HIV-1 gene expression but validates and improves the specificity of activating a specific anti-HIV-1 inhibitory signal in the host cell. Omics analyses revealed less than a 0.7% of host processes were changed by 5342191. Future evaluation of this compound or its derivatives in an in vivo model of HIV-1 infection will be invaluable for confirming our findings. This study supports the future targeting of host intracellular signaling as a new method for managing HIV-1 infection.