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

We have demonstrated that the formation of an upstream attenuator hairpin can be controlled by alternate basepairing schemes to achieve −1 PRF activity regulation (31) . was designed to form either 19 or 20 bp with its complementary upstream RNA target to generate RNA-DNA duplexes of similar stability (37) . We then measured the effect of each antisense on attenuation efficiency of a −1 PRF reporter (6BPGC-SARSPK) containing the upstream 6BPGC attenuator hairpin and a downstream SARS-CoV −1 PRF pseudoknot stimulator (30) . The −1 PRF attenuation was tracked by decreased −1 PRF efficiency, which was observed from decreased −1 frame or/and increased 0 frame translation products upon antisense addition in a −1 PRF assay. Interestingly, addition of 6BPGC-5 -DNA resulted in a dose-dependent loss of 6BPGC attenuator activity consistent with antisense-mediated attenuation hairpin disruption, whereas addition of 6BPGC-3 -DNA did not suppress attenuator activity of 6BPGC ( Figure 1A and B). This means that −1 PRF activity can be oppositely controlled in-trans by antisense DNA oligonucleotides designed to target either side of the stem region of an upstream attenuator hairpin, providing a way to sequence-specifically regulate −1 PRF.