A general strategy to inhibiting viral −1 frameshifting based on upstream attenuation duplex formation

Viral -1 programmed ribosomal frameshifting (PRF) as a potential antiviral target has attracted interest because many human viral pathogens, including human immunodeficiency virus (HIV) and coronaviruses, rely on -1 PRF for optimal propagation. Efficient eukaryotic -1 PRF requires an optimally placed stimulator structure downstream of the frameshifting site and different strategies targeting viral -1 PRF stimulators have been developed. However, accessing particular -1 PRF stimulator information represents a bottle-neck in combating the emerging epidemic viral pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV). Recently, an RNA hairpin upstream of frameshifting site was shown to act as a cis-element to attenuate -1 PRF with mechanism unknown. Here, we show that an upstream duplex formed in-trans, by annealing an antisense to its complementary mRNA sequence upstream of frameshifting site, can replace an upstream hairpin to attenuate -1 PRF efficiently. This finding indicates that the formation of a proximal upstream duplex is the main determining factor responsible for -1 PRF attenuation and provides mechanistic insight. Additionally, the antisense-mediated upstream duplex approach downregulates -1 PRF stimulated by distinct -1 PRF stimulators, including those of MERS-CoV, suggesting its general application potential as a robust means to evaluating viral -1 PRF inhibition as soon as the sequence information of an emerging human coronavirus is available.