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

An antisense approach has been applied to disrupt the downstream −1 PRF stimulator of SARS-CoV (26) . However, the success of this approach requires characterization of the boundary of the downstream stimulator. Such information may not be available for a new outbreak pathogen, such as the MERS-CoV (27) . Indeed, we found that both a SARS-CoV type 3-stems pseudoknot (28, (44) (45) (46) and a 229E type kissing hairpin pseudoknot (47) might exist downstream of the UUUAAAC −1 PRF slippery site of the MERS-CoV (48) (Supplementary Figure S5A and B) . Additionally, two −1 PRF reporters containing sequence deletions of MERS-CoV −1 PRF signal that block the formation of either pseudoknot (Supplementary Figure S6 ) possessed substantial −1 PRF activity both in vitro and in 293T cell ( Figure 5 A-D) , suggesting that both pseudoknots may function during viral replication although the contribution of either pseudoknot to viral −1 PRF activity requires further study. 2 OMe-modified RNA was used to evaluate the viral −1 PRF attenuation activity of the antisense-mediated upstream duplex in human cell lysate because DNA antisense would be digested in crude cellular lysates. We found that a 2 OMe-modified antisense RNA oligonucleotide capable of mediating upstream duplex formation (Supplementary Figure S6A ) efficiently downregulated the in vitro −1 PRF activities of the MERS-CoV viral sequence capable of forming alternative downstream stimulators in a dosage-dependent manner in human 293T cell lysate, whereas the attenuation effect was neutralized by the co-existence of an anti-antisense (Supplementary Figure S6A and Figure 6A) . A more rigorous statistical analysis recommended for p2-luc based assay (36) using 10 M of 2 OMe-modified antisense RNA and a read-through control led to a similar extent of inhibition ( Figure 6B ). Therefore, targeting the sequence upstream of the slippery site represents an efficient and straightforward approach to viral −1 PRF inhibition because detailed knowledge of the downstream stimulator boundary is not required. This approach should be useful in looking for quick therapeutic solutions to emerging pathogens such as the MERS-CoV.