Cucumber green mottle mosaic virus encodes additional small proteins with specific subcellular localizations and virulence function.

The vast majority of known viruses belong to the positive-sense single-stranded RNA (+ssRNA) class. Tobamoviruses are among the most destructive plant viruses and threaten global food security. It is generally accepted that +ssRNA viruses including tobamoviruses encode proteins solely on their positive strand (+RNA). Here, we identified additional open-reading frames (ORFs) in the negative strand of tobamoviruses, named reverse ORFs (rORFs). Using cucumber green mottle mosaic virus (CGMMV) as a model, we detected the corresponding peptides of rORFs by mass spectrometry analysis and confirmed the translation of rORFs by ribosome profiling. Furthermore, we demonstrated that these rORFs may be translated from an internal ribosome entry site. Mutation of rORF1 and rORF2 significantly reduced the virulence of CGMMV, whereas ectopic expression of rORF1 and rORF2 could rescue the pathogenicity of the mutants. While the rORF2 protein localizes at the cell membrane and in the nucleolus, rORF1 colocalizes with peroxisomes, where it interacts with the viral 126-kD replication protein. Additionally, we screened peroxisomal rORF1-interacting proteins using artificial intelligence tools and found that PEX3 mediated rORF1 targeting to peroxisomes. This study reveals that the tobamoviral proteome is larger than previously thought, and sheds light on peroxisomes as novel virulence targets important for virus infectivity.