Diversity of RNA Virus Populations Is Increased in the Presence of Engineered Synonymous Mutations That Disrupt RNA Structural Elements

Previous studies have shown that engineered mutants of cucumber mosaic virus (CMV) with synonymous changes in the coat protein (CP) open reading frame (ORF), accumulate de novo amino acid (aa) substitutions in the CP when passaged in planta. To explore this phenomenon further, we applied whole-genome sequencing to assess the repeatability and extent of these de novo substitutions. Infectious mutants were engineered to contain synonymous changes in one of four ORF stem-loop (SL) structures, SL1362, SL1439, SL1745 and SL1816, or in a highly conserved structure, SL2073, in the 3′ untranslated region. These changes occurred either in the loop or stem, thereby either maintaining or disrupting the predicted SL structure. The mutants were inoculated and then passaged on plants of Nicotiana tabacum, after which the full-length viral sequence (8 kb) was sequenced by an amplicon tiling approach using the Illumina Nextera platform. Across the whole genome, a statistically significant increase (all mutants vs. all wild-type) in de novo single nucleotide polymorphisms (synonymous and non-synonymous) occurred across the mutants as minor (≥10%) and dominant populations, which is suggestive of compensatory genome-wide epistatic changes induced by changes in SLs. Importantly, the results corroborated previous findings by confirming the repeated emergence of substitution P77L in the CP, while also identifying novel substitutions, particularly in the replication-associated 1a protein. This study provides additional genome-wide evidence on a newly described phenomenon, paving the way for additional studies on the function of these de novo changes.