The Highly Conserved Codon following the Slippery Sequence Supports -1 Frameshift Efficiency at the HIV-1 Frameshift Site

Mathew, Suneeth F.; Crowe-McAuliffe, Caillan; Graves, Ryan; Cardno, Tony S.; McKinney, Cushla; Poole, Elizabeth S.; Tate, Warren P.

Cell culture COS-7 or HEK293T cells (obtained from the American Type Culture Collection) were maintained in DMEM (Invitrogen) containing 10% (v/v) fetal bovine serum (Invitrogen). For transfection of the dual luciferase vectors, cells were harvested at 80% confluence and seeded into 24-well plates at a density of 2 or 4 × 10 4 cells/well (COS-7 and HEK293T respectively), and grown until 60% confluent. Vectors were transfected using the FuGENE 6 Transfection Reagent (Roche) at a ratio of 3 μl to 1 μg DNA. The reporter vector was transfected at 0.25 μg; and eRF1, shRNA and suppressor tRNA vectors up to 1 μg per well. For bicistronic fluorophore vectors, 1 × 10 5 cells/well were seeded into 24-well plates and transfection of 0.5 μg reporter vector was performed at the same time as seeding (ratio FuGENE 6: DNA; 4 μl: 1 μg). Cells were incubated for 48 h after transfection of eRF1 and suppressor tRNA vectors, or 120 h after transfection of shRNA vectors with media changes at 60 h and 90 h, then washed twice with phosphate buffered saline and lysed with 100 μl Passive Lysis Buffer (Promega). assayed as described [46] . Lysate (10 μl) was mixed with RLuc or Luc + assay buffer (50 μl) and relative light units were measured using an AutoLumat LB953 (EG&G Berthold). Relative frameshift efficiencies were calculated as: (Luc +