Sequence variability of helper component protein of potato virus Y identified by thermodynamic methods

An extent of helper component protein (HC-Pro) sequence variability within virus population of single Czech isolate of potato virus Y-NTN (pVY(NTN)) Nicola was identified by temperature-gradient gel electrophoresis (TGGE) and heteroduplex analysis. HC-Pro region was approximated with 6 pairs of primers derived from Hungarian pVY(NTN) isolate (sequence AC M95491). Immunocapture reverse transcription - polymerase chain reaction (RT-PCR) was used to obtain six mixtures of individual overlapping polymerase chain reaction (PCR) products. cDNA libraries were prepared by cloning of purified PCR products in pCR-Script vector and screened by heteroduplex analyses. 15 different subfragments within the HC-Pro region were isolated and sequenced. In comparison to AC M95491, 19 nucleotide changes were identified, 13 led to amino acid (aa) changes. In comparison to available post-transcriptional gene silencing (PTGS) specific suppressor HC-Pro sequences of potyviruses we found out 4 aa changes in conserved regions.