Medicinal chemistry optimization of a previously described stilbene inhibitor of HIV-1, 5350150 (2-(2-(5-nitro-2-thienyl)vinyl)quinoline), led to the identification of the thiazole-5-carboxamide derivative (GPS491), which retained potent anti-HIV-1 activity with reduced toxicity. In this report, we demonstrate that the block of HIV-1 replication by GPS491 is accompanied by a drastic inhibition of viral gene expression (IC₅₀ ~ 0.25 µM), and alterations in the production of unspliced, singly spliced, and multiply spliced HIV-1 RNAs. GPS491 also inhibited the replication of adenovirus and multiple coronaviruses. Low µM doses of GPS491 reduced adenovirus infectious yield ~1000 fold, altered virus early gene expression/viral E1A RNA processing, blocked viral DNA amplification, and inhibited late (hexon) gene expression. Loss of replication of multiple coronaviruses (229E, OC43, SARS-CoV2) upon GPS491 addition was associated with the inhibition of viral structural protein expression and the formation of virus particles. Consistent with the observed changes in viral RNA processing, GPS491 treatment induced selective alterations in the accumulation/phosphorylation/function of splicing regulatory SR proteins. Our study establishes that a compound that impacts the activity of cellular factors involved in RNA processing can prevent the replication of several viruses with minimal effect on cell viability.

RNA processing plays a key role in gene expression, allowing for increased protein diversity and functional complexity. Consequently, modulating RNA processing can impact gene function. Given HIV-1’s reliance on host RNA processing machinery for viral protein production/replication, modulators of this process could serve as novel anti-virals to complement and/or enhance existing therapies. In this study, screening of several serine-arginine-rich (SR) kinase inhibitors for their impact on HIV-1 gene expression identified harmine as an inhibitor of HIV-1 gene expression in several cell lines and primary CD4 ⁺ T cells/macrophages at low micromolar concentrations with limited cell toxicity. Harmine induced a loss of viral structural protein expression associated with reduced HIV-1 unspliced and singly-spliced HIV-1 RNA levels but limited impact on multiply spliced RNAs. Although harmine is a known inhibitor of both DYRK1A and monoaminoxidase A (MAO A), neither DYRK1A depletion nor other MAO A inhibitors had any effect on HIV-1 expression. However, the compound altered the expression of several other SR kinases in primary CD4 ⁺ T cells, increasing CLK1 and reducing CLK2 kinase levels, effects known to regulate HIV-1 expression. Harmine was also unique among the SR kinase inhibitors tested for its ability to suppress replication of a seasonal coronavirus, human coronavirus (HCoV)-229E, and multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, reducing viral protein expression and virus release. Harmine acts post-entry, arresting virus replication even after the onset of viral protein production. At doses required to suppress HIV-1 replication, harmine had limited impact on the host transcriptome, alternative splicing, or alterative polyadenylation as assessed by RNA-Seq. Together, our study demonstrates the feasibility of targeting host RNA processing to inhibit a range of viruses with minimal impact on the host cell. IMPORTANCE: This study highlights the crucial role RNA processing plays in regulating viral gene expression and replication. By targeting SR kinases, we identified harmine as a potent inhibitor of HIV-1 as well as coronavirus (HCoV-229E and multiple SARS-CoV-2 variants) replication. Harmine inhibits HIV-1 protein expression and reduces accumulation of HIV-1 RNAs in both cell lines and primary CD4 ⁺ T cells. Harmine also suppresses coronavirus replication post-viral entry by preferentially reducing coronavirus sub-genomic RNA accumulation. By focusing on host factors rather than viral targets, our study offers a novel approach to combating viral infections that is effective against a range of unrelated viruses. Moreover, at doses required to inhibit virus replication, harmine had limited toxicity and minimal effect on the host transcriptome. These findings support the viability of targeting host cellular processes as a means of developing broad-spectrum anti-virals.

Treatment options for viral infections are limited and viruses have proven adept at evolving resistance to many existing therapies, highlighting a significant vulnerability in our defenses. In response to this challenge, we explored the modulation of cellular RNA metabolic processes as an alternative paradigm to antiviral development. Previously, the small molecule 5342191 was identified as a potent inhibitor of HIV-1 replication by altering viral RNA accumulation at doses that minimally affect host gene expression. In this report, we document 5342191 as a potent inhibitor of adenovirus, coronavirus, and influenza replication. In each case, 5342191-mediated reduction in virus replication was associated with altered viral RNA accumulation and loss of viral structural protein expression. Interestingly, while resistant viruses were rapidly isolated for compounds targeting either virus-encoded proteases or polymerases, we have not yet isolated 5342191-resistant variants of coronavirus or influenza. As with HIV-1, 5342191’s inhibition of coronaviruses and influenza is mediated through the activation of specific cell signaling networks, including GPCR and/or MAPK signaling pathways that ultimately affect SR kinase expression. Together, these studies highlight the therapeutic potential of compounds that target cellular processes essential for the replication of multiple viruses. Not only do these compounds hold promise as broad-spectrum antivirals, but they also offer the potential of greater resilience in combating viral infections.