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

Capped viral −1 PRF reporter mRNAs were prepared using a mMESSAGE mMACHINE high-yield capped RNA transcription kit (Ambion) by following manufacturer's instructions. Reticulocyte lysate (Ambion) as well as human 293T cell lysate was used to generate shifted and non-shifted protein products for frameshifting analysis. For radioactivity-based assay in reticulocyte lysate, a total of 5 l reaction containing 100 ng of capped reporter mRNA, 2.5 l of translation lysate and 0.2 l of 10 Ci/l 35 Slabeled methionine (NEN) was incubated at 30 • C for 1.5-2 h The in vitro translation samples were resolved by 12% sodium dodecylsulphate-polyacrylamide gel electrophoresis and exposed to a phosphorimager screen for quantification after drying. Frameshifting efficiencies were calculated, by dividing the counts of the shifted product by the sum of the counts for both shifted and non-shifted products, with calibration of the methionine content in each protein product. We presented all of our radioactivity-based in vitro −1 PRF results in term of relative −1 PRF activity and the ribosome drop-off effect (30) was removed by this procedure. In vitro dual-luciferase based −1 PRF assay was performed in reticulocyte lysate or human 293T cell lysate as described in each experiment. For a 10 l in vitro translation reaction using 293T cell lysate, the reaction contained ∼5 g/l cell lysate and 500 ng of RNA templates in a translation buffer of 20 mM HEPES, pH7.6, 80 mM potassium acetate, 1 mM Magnesium acetate, 1 mM ATP, 0.12 mM GTP, 20 mM creatine-phosphate, 0.1 mg/ml creatine phosphokinase, 2 mM DTT, 0.15 mM spermidine and 400 U/ml RNasin (Promega) (35) . The reactions were incubated at 30 • C for 2 h. Luciferase activity measurements were performed using the Dual Luciferase TM reporter assay (Promega) according to the manufacturer's instructions on a CHAMELEON TM multi-label plate reader (HIDEX). Frameshifting efficiency was then calculated according to previously described procedures with read-through controls (30) .