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Studies of the Astrovirus Signal That Induces (−1) Ribosomal Frameshifting

  • Terry L. Lewis
  • Suzanne M. Matsui
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 412)

Summary

Human astroviruses use a (−1) ribosomal frameshift mechanism to regulate expression of the viral RNA-dependent RNA polymerase gene. To evaluate the efficiency of the astrovirus frameshift signal in cell culture, different regions of the frameshift signal were cloned into the rhesus rotavirus VP4 gene and expressed in an infection-transfection transient expression cell culture system. The various astrovirus-VP4 constructs were transfected into BHK-21 cells infected with a recombinant vaccinia virus that expresses T7 RNA polymerase (vTF7—3). All constructs contain a T7 promoter, a picornavirus internal ribosome entry site, and a T7 terminator. Frameshifted and non-frameshifted proteins were distinguished by immunoprecipitation with monoclonal antibodies specific for either the VP4 amino-or carboxy-terminus. Frameshifting efficiency was calculated as the ratio of radioactive counts in the frameshifted protein to the total counts in both the frameshifted and nonframeshifted proteins as determined by Phosphorimager analysis. We found the efficiency of astrovirus frameshifting in intact cells to be 25–28%, significantly greater than the 5–7% efficiency reported previously in a cell-free uncoupled translation system. Since the transfected plasmid is expressed in the cytoplasm in the infection-transfection system, the frameshifting efficiency determined by this assay may be a more accurate reflection of the level of frameshifting for this RNA virus in which transcription and translation are likely coupled in the cytoplasm of infected cells. This hypothesis is supported by the observation that the level of astrovirus frameshifting is increased three-fold when evaluated in a cell-free coupled transcription-translation system. These studies also confirm in intact cells what was previously determined in cell-free studies: the shifty hep-tamer is an absolute requirement for astrovirus ribosomal frameshifting, but deletion of sequences downstream of the stem-loop that are potentially involved in pseudoknot formation does not affect the efficiency of frameshifting.

Keywords

Intact Cell Recombinant Vaccinia Virus Ribosomal Frameshifting Human Astroviruses Frameshift Signal 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1997

Authors and Affiliations

  • Terry L. Lewis
    • 1
    • 2
  • Suzanne M. Matsui
    • 1
    • 2
    • 3
  1. 1.Program in Cancer BiologyUSA
  2. 2.Department of Medicine (Gastroenterology)Stanford University School of MedicineStanfordUSA
  3. 3.VA Palo Alto Health Care System 154RPalo AltoUSA

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