Fig 9.

Mutational analysis of the RCAS frameshifting signal. (A) Mutations in pMV-RCAS are shown, with pseudoknot mutations color coded as described for Fig. 2A. The PK2 and PK4 regions, which form stem 2* of the pseudoknot, are in bold. The stop codon of the gag ORF is in red. (B) Ribosomal frameshift assays of pMV-RCAS variants were carried out in RRL as described in the legend to Fig. 2. Molecular sizes (in kDa) are indicated on the left. (C) DF1 cells were transfected with pRCAS-WT or mutant derivatives, and released virus was used subsequently to infect new cells. Cell lysates (upper panel) and supernatant virus (lower panel) were analyzed by SDS-PAGE (8% and 15% gels, respectively) and Western blotting using a polyclonal anti-CA (p27) serum. (D) Reverse transcriptase activities were assayed as described in Materials and Methods and quantified by scintillation counting.