Mutational study reveals that tertiary interactions are conserved in ribosomal frameshifting pseudoknots of two luteoviruses

Expression of the putative replicase of potato leafroll virus (PLRV) is reg ulated by -1 ribosomal frameshifting in which a primary viral transcript ha s two overlapping open reading frames (ORFs). A region of 39 nt at the junc tion of the two ORFs is essential for frameshifting to occur. It has been s hown to harbor two signals, one active on the level of the primary structur e, termed the slippery sequence, and one component that forms a secondary o r tertiary level structure, described as either a pseudoknot or a stem-loop motif. We have performed extensive site-directed mutagenesis of the frames hifting region and analyzed individual mutants for their ability to promote -1 frameshifting in vitro. Detailed comparison of our results with analogo us mutants in the frameshifting region of the evolutionarily related beet w estern yellow virus, for which a crystal structure is available, unequivoca lly argues for the pseudoknot to be the structural motif necessary for the frameshifting function in PLRV transcripts. Mutations in PLRV that affect p utative pseudoknot-specific tertiary-base interactions drastically affect f rameshifting activity. In addition, a specific deletion mutant was identifi ed that displayed PLRV wild-type frameshifting activity with only 22 nt ava ilable for pseudoknot formation.