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Abstract

Continued development of antiviral responses requires more than immunity-based solutions; developing high-throughput cell-based assays to identify small molecule inhibitors against viral translation mechanisms will be essential for antiviral development moving forward. StopGo translation is a unique non-canonical translational event induced by the specific amino acid sequence of the 2A peptide that results in the release of a nascent polypeptide chain and translation downstream without termination or reinitiation. StopGo translation is important for viral replication in several viruses. Despite ample use in biotechnology and current applications in therapeutics, StopGo translation is not entirely understood, and there are no known modulators of the activity. We have developed a novel yeast growth reporter platform to identify chemical inhibitors of StopGo translation. Screening a large chemical library (68,000 compounds) identified 463 validated candidates. Compounds were then screened over several concentrations, validating 102 candidates with a dose-dependent response. Candidates were then evaluated for their ability to inhibit a model virus Cricket Paralysis Virus (CrPV) infection and elicit changes to protein localization in a fluorescent protein subcellular localization reporter. One compound led to decreased viral protein expression. -1 Programmed Ribosomal Frameshifting (-1 PRF) is another non-canonical translation mechanism; -1 PRF is essential for SARS-CoV-2 infection as enzymes critical for replication, such as the RNA-dependent RNA polymerase, are encoded in the -1 frame. As -1 PRF is highly conserved between coronaviruses, it is a strong candidate to target for viral inhibition. We have adapted the yeast growth reporter platform that senses 2A peptide StopGo translation to evaluate compounds capable of inhibiting -1 PRF. Screening of an extensive chemical library (68,000 compounds) identified 519 compounds that may inhibit -1 PRF. Candidates were not validated on this system, however. The development of this high-throughput screening platform lays the foundation of an adaptable cell-based screen that can be rapidly modified to identify inhibitors against viral translation mechanisms. Identification of compounds that inhibit StopGo translation contributes to the tool-kit available for 2A therapeutics and applications in biotechnology and can reveal mechanistic details of StopGo translation.