Investigating -1 PRF in Coronaviruses
Overview. Coronaviruses are positive-strand RNA viruses that represent a significant threat to public health because they have pandemic potential. Antiviral therapies that stop viral replication are key to limiting viral transmission and disease. Steps in the viral life cycle that are both unique to the virus and critical to viral replication represent good antiviral targets. During an early step in the coronavirus life cycle, the RNA genome is translated to produce viral polyproteins. A subset of these proteins, including the RNA dependent RNA polymerase (RdRp), are synthesized by way of a -1 PRF (Figure 5A). This translational event is critical to the coronavirus life cycle because this is how the main replicative enzyme for the virus, the RdRp, is synthesized. Previous research on the SARS coronavirus revealed that altering its -1 PRF efficiency drastically reduced viral replication, suggesting this step is a viable antiviral target. Given that coronavirus frameshift sites are highly-conserved in both sequence and structure, antiviral treatments that target frameshift sites are not likely to readily select for viable drug-resistant virus isolates. Research on -1 PRF in coronaviruses will be important to future drug development efforts targeting this step in the viral life cycle.
Relevant publication:
Swanson Hay, E.G.; Maille, M.M.+; Zafferani, M.; Puray-Chavez, M.; Xia, M.; Luu, T.B. Muralidharan, D.; Li, M.L.; Brewer, G.; Kutluay, S.B.; Mouzakis, K.D.*; Hargrove, A.E.* SARS-CoV-2 Antivirals Identified from Small Molecule Modulators of Programmed –1 Ribosomal Frameshifting. ACS Infectious Diseases 2026, 5 (12). DOI: 10.1021/acsinfecdis.5c00774
Article Highlights: In this work, we aimed to identify small molecules that target the highly conserved SARS-CoV-2 frameshift site pseudoknot. An RNA-biased synthetic small molecule library screen against the RNA pseudoknot structure identified several candidate molecules. Of these, seven showed evidence of dose-dependent activity in in vitro and in cellulo frameshift assays and inhibited SARS-CoV-2 replication in cell culture. While binding was required for activity, affinity did not correlate with frameshift inhibition. Similarly, small molecule impacts to pseudoknot stability, as measured with an RT-qPCR assay, did not predict impacts on the frameshift efficiency. Comparison of the hits, as well as previously published inhibitors, across several assays supports the need for multiple characterization methods in small molecule–RNA targeting and the possibility of multiple modes of action for –1 PRF modulation. This work underscores the complexity of targeting dynamic RNA structures and the benefits of screening early for function and later optimizing for mode of action. The use of orthogonal techniques led to the identification of several promising hits and a lead antiviral compound.
Impact: Identified seven small molecules that target the SARS-CoV-2 frameshift pseudoknot, decrease its –1 PRF efficiency, and have antiviral activity.
Ongoing projects:
- Measuring the frameshift efficiency of the SARS-CoV-2 frameshift site using dual fluorescence reporter proteins
