RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Ishimaru, Daniella; Plant, Ewan P.; Sims, Amy C.; Yount, Boyd L.; Roth, Braden M.; Eldho, Nadukkudy V.; Pérez-Alvarado, Gabriela C.; Armbruster, David W.; Baric, Ralph S.; Dinman, Jonathan D.; Taylor, Deborah R.; Hennig, Mirko

Transfected VeroE6 cells were grown overnight in Dulbecco's Modified Eagle Medium supplemented with 10% Fetal Bovine Serum at 37 C. Cells were disrupted using the passive lysis buffer (Promega). Luminescence reactions were initiated by addition of 10-20 ml of cell lysates to 100 ml of the Promega LAR II buffer and completed by addition of 100 ml Stop-n-Glo reagent. Luminescence was measured using a Turner Design TD20/20. At least three replicates were performed within each assay, and all assays were repeated at least three times until the data were normally distributed (26) . The frequency of frameshifting is expressed as a ratio of firefly to Renilla luciferase from a test plasmid divided by the analogous ratio from the read-through control plasmid multiplied by 100%. Fold change, standard error and estimates of the P-values for ratiometric analyses were performed as previously described (26) .