UC Davis

Two small ORFs were identified through the use of ORF predictors in genomes of two cucurbit-infecting tobamoviruses: Cucumber green mottle mosaic virus (CGMMV) and Watermelon green mottle mosaic virus (WGMMV). One of these ORFs (ORFH) is found in both viruses, while the other ORF (ORFN) is only found in WGMMV. In addition to these two viruses, similar ORFs can also be found in several but not all other cucurbit-infecting tobamoviruses. ORFH overlaps the ORF encoding the 30kDa of CGMMV and WGMMV, but in the +1 reading frame register. Transient expression of ORFH fused to GFP showed fluorescence localization to the endoplasmic reticulum in Nicotiana benthamiana epidermal cells. Truncation mutations of ORFH in CGMMV do not affect the virus’s ability to infect systemically in Nicotiana bethamiana plants, or two melon cultivars Laredo and Expedition. ORFN is located in WGMMV, overlapping the 126 and 183 kDa protein ORFs, also in the +1 reading frame register. The predicted ORFN protein contains a bi-partite nuclear localization signal, which when disrupted changes the nuclear localization to a chloroplast localization. Western blots with an ORFN protein specific antibody show a larger protein than the size predicted for the ORFN protein, suggesting a fusion protein or a protein aggregate. Mutations introduced into the ORFN resulted in mutant WGMMV infections which showed less severe symptoms in N. benthamiana and cucurbit plants, but no changes in viral titer for wildtype and WGMMV mutants were observed. Although it has not yet been confirmed whether these predicted proteins are produced during infection, the presence of these ORFs in more than one virus genome suggests that they are not just the result of prediction errors or artifacts within genome assemblies.