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J. Virol., May 1996, 2869-2875, Vol 70, No. 5
Copyright © 1996, American Society for Microbiology

Astrovirus ribosomal frameshifting in an infection-transfection transient expression system

TL Lewis and SM Matsui
Program in Cancer Biology, Stanford University School of Medicine, California 94305-5487, USA.

Different regions of the human astrovirus frameshift signal were cloned into the rhesus rotavirus VP4 gene and evaluated in an infection- transfection transient expression cell culture system. BHK-21 cells, infected with a vaccinia virus that expresses T7 RNA polymerase (vTF7- 3), were transfected with the various astrovirus-VP4 constructs. All constructs were driven by a T7 promoter and contained an internal ribosome entry site. Frameshifted and nonframeshifted protein products were immunoprecipitated with VP4 amino- and carboxy-terminal-specific monoclonal antibodies, and their ratios were determined by PhosphorImager analysis. The efficiency of frameshifting was 25 to 28%, significantly greater than the 5 to 7% efficiency reported previously in a cell-free translation 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 heptamer was an absolute requirement for frameshifting in both cell-free and intact-cell systems, while deletion of the potential downstream pseudoknot region did not affect the efficiency of frameshifting.


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