Potato virus X (PVX) is widely used as a peptide presentation system in plant biotechnology, mostly by fusion of desired peptides to the N-terminus of its capsid protein (CP). Considering that some epitopes can interfere with the stability and/or self-assembly of PVX CP when fused to its N-terminus, we evaluated four other possible sites for fusion using the E7 epitope derived from human papillomavirus type 16 with different tags. We prepared eight different PVX CP constructs modified with the E7 epitope fused with the 6xHis tag in both orientations (6xHis-E7, E7-6xHis), cloned them into the PVX-based vector pGR106 and expressed them transiently in Nicotiana benthamina plants. Only the fusion site located after amino acid 23 led to systemic infection of plants and the production of recombinant proteins, but no viral particles were detected. When we replaced the 6xHis with StrepII tag, the modified virus infected plants systemically, expressed proteins assembled into viral particles and the epitopes were located on the particle surface. The results of this study indicate that the new position within the PVX CP can be used for peptide presentation on the surface of PVX particles and is promising for PVX based production of therapeutic compounds in plants.
The sequences of the 3′‐terminal region of four Czech Potato virus M isolates VIRUBRA 4/007, VIRUBRA 4/009, VIRUBRA 4/016 and VIRUBRA 4/035 were determined and compared with sequences of PVM isolates available in GenBank. Among the Czech isolates, VIRUBRA 4/007 and 4/016 as well as VIRUBRA 4/016 and 4/035 showed the highest nucleotide identity (93%). Isolates VIRUBRA 4/007, 4/016 and 4/035 were most similar to the PV0273 isolate from Germany and to the wild isolate from Russia. Interestingly, isolate VIRUBRA 4/009 significantly differed from the other three Czech isolates and was the only European isolate that showed the highest nucleotide identity with American isolates. Moreover, the PVM isolates from the Czech Republic and Germany differed in their host range. Phylogenetic analysis based on ORF5 coding for coat protein showed that the Czech isolates could be classified in two of the three groupings of the phylogenetic tree obtained. This is the first report on molecular and biological analysis of the genome sequences of PVM isolates from the Czech Republic.
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