The pokeweed antiviral protein specifically inhibits Ty1-directed +1 ribosomal frameshifting and retrotransposition in Saccharomyces cerevisiae

Abstract

Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to produce Gag-Pol fusion proteins. The efficiency of these frameshift events determines the ratio of viral Gag to Gag-Pol proteins available for viral particle morphogenesis, and changes in ribosomal frameshift efficiencies can severely inhibit virus propagation. Since ribosomal frameshifting occurs during the elongation phase of protein translation, it is reasonable to hypothesize that agents that affect the different steps in this process may also have an impact on programmed ribosomal frameshifting. We examined the molecular mechanisms governing programmed ribosomal frameshifting by using two viruses of the yeast Saccharomyces cerevisiae. Here, we present evidence that pokeweed antiviral protein (PAP), a single-chain ribosomal inhibitory protein that depurinates an adenine residue in the alpha-sarcin loop of 25S rRNA and inhibits translocation, specifically inhibits Ty1-directed +1 ribosomal frameshifting in intact yeast cells and in an in vitro assay system. Using an in vivo assay for Ty1 retrotransposition, we show that PAP specifically inhibits Ty1 retrotransposition, suggesting that Ty1 viral particle morphogenesis is inhibited in infected cells. PAP does not affect programmed -1 ribosomal frameshift efficiencies, nor does it have a noticeable impact on the ability of cells to maintain the M1-dependent killer virus phenotype, suggesting that -1 ribosomal frameshifting does not occur after the peptidyltransferase reaction. These results provide the first evidence that PAP has viral RNA-specific effects in vivo which may be responsible for the mechanism of its antiviral activity.

FIG. 1

Vectors used to measure programmed ribosomal frameshifting efficiencies in vivo (A) and in vitro (B). The in vivo 0 frame control reporter plasmid pT125 and the −1 ribosomal frameshift test plasmid pF8 are described elsewhere (12). The +1 ribosomal frameshift test plasmid pJD104 is described elsewhere (2). In these constructs, transcription is driven from the constitutive phosphoglycerol kinase 1 (PGK1) promoter. The in vitro 0 frame control plasmid pLUC0 and the −1 ribosomal frameshift test plasmid pJD120 are described elsewhere (14). Construction of the in vitro +1 ribosomal frameshift plasmid pJD120.104 is described in Materials and Methods. The efficiencies of programmed ribosomal frameshifting were determined by dividing the enzymatic activities produced from the frameshift reporters (−1 or +1) by the enzymatic activities produced from the 0 frame controls and multiplying the resulting ratios by 100. The approximate locations of −1, +1, and 0 frame termination codons are indicated by numbers.

FIG. 2

Effects of PAP on programmed ribosomal frameshifting in vivo. Cultures of yeast cells harboring frameshift indicator vectors (0 frame control and −1 or +1 ribosomal frameshift test vectors) and either pNT123 (galactose-inducible wild-type PAP), pNT123-2 (galactose-inducible active-site mutant PAP), or no other vector were split into selective media containing either 2% galactose (induced) or 2% raffinose (uninduced) and incubated for 5 h. β-Galactosidase activities were then determined. The efficiencies of programmed ribosomal frameshifting are taken from Table 1. To obtain the percent of control, the ratios of frameshifting in PAP-induced and uninduced cells (Gal/Raf in pNT123 and pNT123-2; Table 1) were normalized to those in control cells (Gal/Raf in PSY1; Table 1). RFS, ribosomal frameshift.

FIG. 3

Effects of PAP in vitro on −1 and +1 ribosomal frameshifting (RFS) efficiencies. Translation-competent rabbit reticulocyte lysates were preincubated at 28°C for 15 min with the indicated amounts of purified PAP. Then, 20 ng of each of the synthetic luciferase reporter mRNAs (0 frame control and −1 or +1 ribosomal frameshift test mRNAs) was added to the PAP-lysate mixture and incubated at 28°C for 45 min. Luciferase activities were then determined by luminometry. Ribosomal frameshifting efficiencies were calculated by determining the ratios of luciferase activities produced by the test mRNAs to the 0 frame control value. Percent of control represents the efficiency of ribosomal frameshifting plotted as a percentage of that in the no-PAP control.

FIG. 4

PAP inhibits Ty1 retrotransposition. PSY1 cells were transformed with galactose-inducible PAP (pNT188) or vector (YEp351) and pTy1HIS3AI. Cells were grown at 24°C on medium containing 2% galactose but lacking uracil and leucine for 4 days, replica plated at 30°C to medium containing 2% dextrose but lacking uracil and leucine, and subsequently replica plated to medium containing 2% dextrose but lacking histidine. Growth of the colonies was indicative of Ty1 retrotransposition.