Evaluating the Negative‐Strand Coding Potential in Plum Pox Virus

Recent studies proposed that the negative strands of some single-stranded, positive-sense RNA viruses contain reverse open reading frames (rORFs) that encode functional peptides. Highly conserved rORFs were found in the negative strand of viruses within the Potyviridae family, including turnip mosaic virus (TuMV), where rORF2 was shown to be essential for infection and survival. In this study, we analysed the existence and potential functionality of rORFs in plum pox virus (PPV) using mass spectrometry and mutagenesis. Unbiased mass spectrometry analyses failed to identify any rORF-encoded peptides and PPV mutants abolishing the expression of individual rORF-derived peptides showed no significant difference in infection rates compared to the wild-type virus in the experimental host Nicotiana benthamiana or in the natural host Prunus persica. Furthermore, competitive infection experiments revealed that neither the wild type nor the rORF2 knockout mutant, which had proven lethal when tested in TuMV, exhibited any competitive advantage over the other. Taken together, these findings suggest that rORFs in PPV are either not produced or are functionally irrelevant for infection, and they highlight the need to evaluate the role of rORFs in other potyviruses on a case-by-case basis.