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

To explore the potential of upstream duplex-mediated −1 PRF attenuation in anti-viral applications in human coronavirus (hCoV), we analyzed sequences upstream of the slippery sites of six known hCoVs and found a potential hairpin stem upstream of the −1 PRF slippery site of 229E-CoV in addition to the one characterized in SARS-CoV (Supplementary Figure S3A and B) . However, the −1 PRF activity of a shortened p2luc reporter containing the 229E upstream hairpin and an SARS-CoV pseudoknot was not attenuated as efficiently as that by the SARS-CoV attenuator hairpin (Supplementary Figure S4A-C) , and could be explained by the less stable free energy of the 229E upstream hairpin predicted by Mfold (43) . We then asked if an antisense-mediated duplex can compete with the predicted 229E upstream hairpin to generate a potent −1 PRF attenuator in a duplex form. EMSA experiments indicated an isolated 229E upstream RNA hairpin (Supplementary Figure S4D ) forming a complex with an antisense DNA (anti-229E) that targets the 3 -side sequences of the hairpin (Supplementary Figure S4E) , suggesting the formation of an RNA-DNA duplex ( Figure 4A) . Furthermore, the addition of anti-229E led to higher −1 PRF attenuation activity than the cis-formed 229E viral attenuator hairpin alone in a full-length p2luc-based −1 PRF reporter (Supplementary Figure S4E and S4B) . Thus, pre-existing local conformations upstream of −1 PRF slippery site can be programmed by the antisense approach to generate a functional −1 PRF attenuator in the form of a trans-formed duplex.