Single-nucleotide polymorphisms and reading frame shifts in RNA2 recombinant regions of tobacco rattle virus isolates Slu24 and Deb57.

Two previously sequenced tobacco rattle virus (TRV) isolates, Slu24 and Deb57, from Polish potato fields have recombinant RNA2 species. The 3'-proximal region of the Slu24 RNA2 is derived from the 3' terminus of its supporting RNA1, while that of the Deb57 RNA2 is derived from the 3' terminus of the unrelated RNA1 from the isolate SYM or PpK20. Gene structure annotation revealed open reading frames encoding truncated 16-kDa proteins in the recombinant regions of the RNA2 of Deb57 and Slu24. Reading frame shifts, single nucleotide substitutions and deletions occurred during recombination, including shifts from a stop codon or replacements of an internal stop codon. In the recombinant region of the Deb57 RNA2, the first reading frameshift event starts from the AUG start codon of the truncated 16-kDa protein. The second frameshift event, caused by a single nucleotide deletion upstream of the stop codon, leads to the splitting of the stop codon into two amino acid codons and the continuation of translation. In addition, a U-to-A substitution changes a potential internal stop codon UAA, which is caused by recombination-related frame shifts, into the codon AAA, encoding lysine. The replacement of this internal stop codon with an amino acid codon prevented the premature translation termination of the truncated 16-kDa protein. These recombination-related reading frame shifts are the driving force underlying the major differences in the translated amino acids, consequently leading to their translation into distinct polypeptides. Conversely, single nucleotide substitutions in the recombinant regions of the RNA2 of Deb57 or Slu24 resulted in only minor changes in the translated amino acids.