A rapid, inexpensive yeast-based dual-fluorescence assay of programmed—1 ribosomal frameshifting for high-throughput screening

摘要

Programmed ?1 ribosomal frameshifting (?1 PRF) is a mechanism that directs elongating ribosomes to shift-reading frame by 1 base in the 5′ direction that is utilized by many RNA viruses. Importantly, rates of ?1 PRF are fine-tuned by viruses, including Retroviruses, Coronaviruses, Flavivriuses and in two endogenous viruses of the yeast Saccharomyces cerevisiae, to deliver the correct ratios of different viral proteins for efficient replication. Thus, ?1 PRF presents a novel target for antiviral therapeutics. The underlying molecular mechanism of ?1 PRF is conserved from yeast to mammals, enabling yeast to be used as a logical platform for high-throughput screens. Our understanding of the strengths and pitfalls of assays to monitor ?1 PRF have evolved since the initial discovery of ?1 PRF. These include controlling for the effects of drugs on protein expression and mRNA stability, as well as minimizing costs and the requirement for multiple processing steps. Here we describe the development of an automated yeast-based dual fluorescence assay of ?1 PRF that provides a rapid, inexpensive automated pipeline to screen for compounds that alter rates of ?1 PRF which will help to pave the way toward the discovery and development of novel antiviral therapeutics.