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

Hu, Hao-Teng; Cho, Che-Pei; Lin, Ya-Hui; Chang, Kung-Yao

Recently, an RNA hairpin upstream of the −1 frameshifting site of the SARS-CoV has been shown to attenuate −1 PRF depending on hairpin stability and an optimal spacer length between the slippery site and hairpin (28, 29) . This unique upstream hairpin represents the first cis-element capable of downregulating eukaryotic −1 PRF activity and understanding its functional mechanism should provide insight into the mechanism of −1 PRF regulation with antiviral application potential. As the upstream attenuation hairpin is unwound by the ribosome before the ribosome encounters the downstream stimulator, it has been proposed that the refolding dynamics of the hairpin is responsible for its −1 PRF attenuating activity (29) . Here, we found that an RNA-DNA duplex formed by annealing antisense DNA to its complementary mRNA sequence upstream of a −1 PRF slippery site could attenuate −1 PRF to a similar extent as that of an upstream hairpin attenuator. That the cis-formed upstream hairpin can be replaced by a trans-formed duplex suggests upstream duplex formation is the determining element in −1 PRF attenuation. This finding is reminiscent of frameshifting regulation by SD·anti-SD mediated short upstream duplex in 70S ribosome (17, 18) , providing insight on the functional mechanisms of upstream −1 PRF attenuation in 80S ribosome. Furthermore, we apply this upstream duplex attenuator to counteract several viral −1 PRF signals to demonstrate its general application potential as an alternative −1 PRF inhibition approach. Thus, inhibiting −1 PRF by antisense-mediated upstream duplex provides a potentially quick antiviral solution to the emerging highly pathogenic coronaviruses and an opportunity to sequencespecifically regulate −1 PRF related cellular events.