Sequence Element Required for Efficient −1 Ribosomal Frameshifting in Red Clover Necrotic Mosaic Dianthovirus

Abstract

The RNA−1 of the bipartite red clover necrotic mosaic dianthovirus (RCNMV) genome encodes the 88-kDa polymerase. The polymerase is translated from both 5′ proximal and internal open reading frames by a −1 ribosomal frameshifting event. A shifty heptanucleotide conforming to the simultaneous slippage model is identified, and a downstream stem-loop structure and atypical pseudoknot are predicted. A β-glucuronidase reporter assay identified a 118-nucleotide element containing both the shifty heptanucleotide and the predicted secondary structures that were required for efficient −1 ribosomal frameshift expressionin vivo. A series of site-directed and compensatory mutations affecting the base-paired regions of the predicted secondary structure were introduced into a RCNMV RNA−1 cDNA clone from which infectious transcripts were derived. Mutations that destroyed the predicted pseudoknot had no effect on frameshifting efficiencyin vitroor infectivity of the virus, whereas mutations destabilizing the stem-loop structure abolished both ribosomal frameshiftingin vitroand biological activity. These results demonstrate the essential role of a predicted secondary structure that does not involve a pseudoknot in the expression of the RCNMV polymerase by ribosomal frameshifting.