Astrovirus ribosomal frameshifting in an infection-transfection transient expression system.
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.
Lewis
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Program in Cancer Biology, Stanford University School of Medicine, California 94305-5487, USA.
Matsui
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D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D036022:Capsid Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D017934:Coronavirus / Q000235:genetics; D006224:Cricetinae; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000378:metabolism; D016368:Frameshift Mutation; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008253:Macaca mulatta; D001257:Mamastrovirus / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012401:Rotavirus / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014616:Vaccinia virus; D014764:Viral Proteins
D036022:Capsid Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; C060339:VP4 protein, Rotavirus; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases