ASTROVIRUS RIBOSOMAL FRAMESHIFTING IN AN INFECTION TRANSFECTION TRANSIENT EXPRESSION SYSTEM

Different regions of the human astrovirus frameshift signal were clone d into the rhesus rotavirus VP4 gene and evaluated in an infection-tra nsfection transient expression cell culture system. BHK-21 cells, infe cted with a vaccinia virus that expresses T7 RNA polymerase (vTF7-3), were transfected with the various astrovirus-VP4 constructs. All const ructs were driven by a T7 promoter and contained an internal ribosome entry site. Frameshifted and nonframeshifted protein products were imm unoprecipitated with VP4 amino- and carboxy-terminal-specific monoclon al antibodies, and their ratios were determined by PhosphorImager anal ysis. The efficiency of frameshifting was 25 to 28%, significantly gre ater than the 5 to 7% efficiency reported previously in a cell-free tr anslation system. Coupling of transcription and translation in a cell- free system yielded frameshifting efficiencies threefold greater than that of the uncoupled in vitro system. The presence of the shifty hept amer was an absolute requirement for frameshifting in both cell-free a nd intact-cell systems, while deletion of the potential downstream pse udoknot region did not affect the efficiency of 6 frameshifting.