NS1′ of Flaviviruses in the Japanese Encephalitis Virus Serogroup Is a Product of Ribosomal Frameshifting and Plays a Role in Viral Neuroinvasiveness

ABSTRACT Flavivirus NS1 is a nonstructural protein involved in virus replication and regulation of the innate immune response. Interestingly, a larger NS1-related protein, NS1′, is often detected during infection with the members of the Japanese encephalitis virus serogroup of flaviviruses. However, how NS1′ is made and what role it performs in the viral life cycle have not been determined. Here we provide experimental evidence that NS1′ is the product of a −1 ribosomal frameshift event that occurs at a conserved slippery heptanucleotide motif located near the beginning of the NS2A gene and is stimulated by a downstream RNA pseudoknot structure. Using site-directed mutagenesis of these sequence elements in an infectious clone of the Kunjin subtype of West Nile virus, we demonstrate that NS1′ plays a role in viral neuroinvasiveness.

Expression of a Functional Secreted YopN-TyeA Hybrid Protein in Yersinia pestis Is the Result of a +1 Translational Frameshift Event

ABSTRACT YopN is a secreted protein that prior to secretion directly interacts with the cytosolic SycN/YscB chaperone complex and TyeA. This study identifies a secreted YopN-TyeA hybrid protein that is expressed by Yersinia pestis, but not by Yersinia enterocolitica. DNA sequence analysis and site-directed mutagenesis studies demonstrate that the hybrid protein is the result of a +1 translational frameshift event.

Translational bypassing: a new reading alternative of the genetic code

The translation of the genetic code, once thought to be rigid, has been found to be quite flexible, and several alternatives in its reading have been described. An unusual alternative is translational bypassing, a frameshift event where the transition from frame 0 to another frame occurs by translational bypassing of an extended region of the mRNA sequence rather than by slippage past a single nucleotide, as has been described for most examples of frameshifting. Translational bypassing has been characterized in two cases, T4 gene 60 coding for a topoisomerase subunit and in a trpR–lac′Z fusion. The latter was discovered in our laboratory, and the unique bypass mechanism is investigated further in this study. Using a trpR+1–lac′Z fusion system, we show that the Gln codon at the beginning of lacZ end at the 3′ side of the gap is required for bypassing to occur. The Gln codon is part of an mRNA segment that can (potentially) base pair with a segment at the 5′ and of Escherichia coli 16S rRNA. A model of trpR+1–lac′Z bypassing is suggested in which the untranslated region of the mRNA is looped out through base pairing between a segment in the 5′ end of the 16S rRNA and two sites in the mRNA. Translational bypassing is a newly discovered mechanism of gene expression, and trpR is the first cellular gene identified in which such a mechanism could operate. The understanding of this mechanism and its associated signals may be considered a paradigm for the expression of other genes by this alternative reading of the genetic code.Key words: genetic code, translation, frameshifting, trpR.

Upstream stimulators for recoding

Recent progress in elucidation of 5′ stimulatory elements for translational recoding is reviewed. A 5′ Shine–Dalgarno sequence increases both +1 and −1 frameshift efficiency in several genes; examples cited include the E. coli prfB gene encoding release factor 2 and the dnaX gene encoding the γ and τ subunits of DNA polymerase III holoenzyme. The spacing between the Shine–Dalgarno sequence and the shift site is critical in both the +1 and −1 frameshift cassettes; however, the optimal spacing is quite different in the two cases. A frameshift in a mammalian chromosomal gene, ornithine decarboxylase antizyme, has recently been reported; 5′ sequences have been shown to be vital for this frameshift event. Escherichia coli bacteriophage T4 gene 60 encodes a subunit of its type II DNA topoisomerase. The mature gene 60 mRNA contains an internal 50 nucleotide region that appears to be bypassed during translation. A 16 amino acid domain of the nascent peptide is necessary for this bypass to occur.Key words: recoding, frameshifting, peptide factor, stimulatory elements.

Special peptidyl-tRNA molecules can promote translational frameshifting without slippage.

Recently we described an unusual programmed +1 frameshift event in yeast retrotransposon Ty3. Frameshifting depends on the presence of peptidyl-tRNA(AlaCGC) on the GCG codon in the ribosomal P site and on a translational pause stimulated by the slowly decoded AGU codon. Frameshifting occurs on the sequence GCG-AGU-U by out-of-frame binding of a valyl-tRNA to GUU without slippage of peptidyl-tRNA(AlaCGC). This mechanism challenges the conventional understanding that frameshift efficiency must correlate with the ability of mRNA-bound tRNA to slip between cognate or near-cognate codons. Though frameshifting does not require slippery tRNAs, it does require special peptidyl-tRNAs. We show that overproducing a second isoacceptor whose anticodon had been changed to CGC eliminated frameshifting; peptidyl-tRNA(AlaCGC) must have a special capacity to induce +1 frameshifting in the adjacent ribosomal A site. In order to identify other special peptidyl-tRNAs, we tested the ability of each of the other 63 codons to replace GCG in the P site. We found no correlation between the ability to stimulate +1 frameshifting and the ability of the cognate tRNA to slip on the mRNA--several codons predicted to slip efficiently do not stimulate frameshifting, while several predicted not to slip do stimulate frameshifting. By inducing a severe translational pause, we identified eight tRNAs capable of inducing measurable +1 frameshifting, only four of which are predicted to slip on the mRNA. We conclude that in Saccharomyces cerevisiae, special peptidyl-tRNAs can induce frameshifting dependent on some characteristic(s) other than the ability to slip on the mRNA.

Special peptidyl-tRNA molecules can promote translational frameshifting without slippage

Recently we described an unusual programmed +1 frameshift event in yeast retrotransposon Ty3. Frameshifting depends on the presence of peptidyl-tRNA(AlaCGC) on the GCG codon in the ribosomal P site and on a translational pause stimulated by the slowly decoded AGU codon. Frameshifting occurs on the sequence GCG-AGU-U by out-of-frame binding of a valyl-tRNA to GUU without slippage of peptidyl-tRNA(AlaCGC). This mechanism challenges the conventional understanding that frameshift efficiency must correlate with the ability of mRNA-bound tRNA to slip between cognate or near-cognate codons. Though frameshifting does not require slippery tRNAs, it does require special peptidyl-tRNAs. We show that overproducing a second isoacceptor whose anticodon had been changed to CGC eliminated frameshifting; peptidyl-tRNA(AlaCGC) must have a special capacity to induce +1 frameshifting in the adjacent ribosomal A site. In order to identify other special peptidyl-tRNAs, we tested the ability of each of the other 63 codons to replace GCG in the P site. We found no correlation between the ability to stimulate +1 frameshifting and the ability of the cognate tRNA to slip on the mRNA--several codons predicted to slip efficiently do not stimulate frameshifting, while several predicted not to slip do stimulate frameshifting. By inducing a severe translational pause, we identified eight tRNAs capable of inducing measurable +1 frameshifting, only four of which are predicted to slip on the mRNA. We conclude that in Saccharomyces cerevisiae, special peptidyl-tRNAs can induce frameshifting dependent on some characteristic(s) other than the ability to slip on the mRNA.