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

Previously, we have shown that an upstream −1 PRF attenuation hairpin could downregulate −1 PRF stimulated by distinct downstream stimulators (29) . To see if the upstream duplex can be used to attenuate viral −1 PRF stimulators other than that of SARS-CoV, we compared the frameshifting efficiencies of several viral −1 PRF pseudoknot stimulators, including mouse mammary tumor virus (MMTV), simian retrovirus (SRV) and a hairpin stimulator derived from SRV pseudoknot (40) (41) (42) (Supplementary Figure S2) , in the presence of different amount of antisense targeting upstream sequences (Supplementary Figures S2 and S3) . Surprisingly, we found that significant −1 PRF attenuation was observed in the presence of 1 M of antisenses by comparing with those in Figures 1 and 2 . It could be explained by the less stable structures formed upstream of the slippery sites in the current reporters. By contrast, the amount of −1 frame product translated from a read-through control (p2luci) was not affected. Furthermore, the −1 PRF activities can be attenuated to different extents by designed antisense DNA of different complementarities to the sequences upstream of the slippery site. For example, 10 M of restore DNA23 was needed to attenuate −1PRF to similar extent as that of 0.1 M of SRV 5 as ( Figure 3E and F) because restore DNA23 forms less base pairs with the upstream sequences. Taken together, these results indicate that the upstream duplex approach provides a general means of downregulating −1 PRF activity stimulated by distinct types of downstream stimulators in vitro.