Positive Strand RNA viruses persist to pose serious threats to human health and global economies. Disease progression mediated by viral pathogenesis requires numerous intersections between host proteins and viral RNA (vRNA) structures. Host-vRNA complexes drive essential processes in the replication cycles of viruses; as such, they represent untapped targets for therapeutic intervention. In my seminar, I will describe the mechanisms by which the mutually antagonistic human hnRNP A1 and AUF1 proteins compete for the same vRNA structure to differentially regulate Enterovirus (EV) translation efficiency. By screening a library of small molecule RNA binders, we discovered that the compound DMA-135 binds SLII IRES domain to dose-dependently inhibit viral replication by attenuating viral translation. Serial passaging of EV-A71 in the presence of low doses of DMA-135 selects for revertant viruses with drug-resistant mutations that map to the SLII bulge environment. Comparative structure-function studies reveal that the cellular mechanism of action of DMA-135 is to tip the SLII-hnRNP regulatory axis towards significantly lower levels of IRES-dependent translation, and the virus can compensate by evolving mutations that restore homeostasis. Our work defines the antiviral mechanism of action of DMA-135; it demonstrates that functional specificity can be modulated through natural and drug-dependent viral evolution; and it shows how small molecules can reveal new insights about host-virus interfaces that regulate early stages of EV replication.