Valli, A. A.; Domingo-Calap, M. L.; Gonzalez de Pradena, A.; Garcia, J. A.; Cui, H.; Desbiez, C.; Lopez-Moya, J. J.

RNA viruses have evolved sophisticated strategies to exploit the limited encoded information within their typically compact genomes. One of such, named programmed transcriptional slippage (PTS), is defined by the insertion of an additional A at An motifs (n [≥] 6) of newly synthetized viral transcripts to get access to overlapping open reading frames (ORFs). Although key proteins from Ebolavirus and potyvirids (members of the Potyviridae family) are expressed via PTS, available information about this phenomenon is very scarce. Here, by using diverse experimental approaches and a collection of plant/virus combinations, we discover cases in which PTS does not fit with its current definition. In summary, we observe (i) high rate of single nucleotide deletions at slippage motifs, (ii) overlapping ORFs acceded by slippage at an U8 stretch, and (iii) significant changes in slippage rates induced by factors not related to cognate viruses. Moreover, a survey of full-genome sequences from potyvirids shows a widespread occurrence of species-specific An/Un (n [≥] 6) motifs. Even though many of them, but not all, lead to the production of truncated proteins rather than access to overlapping ORFs, these results suggest that slippage motifs appear more frequently than expected and play relevant roles during virus evolution. In conclusion, our data prompt to broaden PTS definition in RNA viruses. Considering the potential of this phenomenon to expand the viral proteome by acceding to overlapping ORFs and/or producing truncated proteins, a revaluation of PTS significance during infections of RNA viruses is required.