A predicted stem-loop in coat protein-coding sequencing of tobacco vein banding mosaic virus is required for efficient replication.

Potyviral Coat protein (CP) is involved in the replication and movement of potyviruses. However, little information is available on the roles of CP-coding sequence in potyviral infection. Here, we introduced synonymous substitutions to the codon c574g575c576 coding conserved residue arginine at position 192 (R192) of tobacco vein banding mosaic virus (TVBMV) CP. Substitution of the codon c574g575c576 to a574g575a576 or a574g575g576, but not c574g575a576, c574g575t576, or c574g575g576, reduced the replication, cell-to-cell movement, and accumulation of TVBMV in Nicotiana benthamiana plants, suggesting that c574 was critical for replication of TVBMV. Nucleotides 531 to 576 of the TVBMV CP-coding sequence were predicted to form a stem-loop structure, in which four consecutive c-g base pairs (C576-G531, c532-g575, c574-g533, and C534-G573) were located at the stem. Synonymous substitutions of R178-codon c532g533c534 to A532G533A534 and A532G533G534, but not c532g533a534, c532g533t534, or c532g533g534, reduced the replication levels, cell-to-cell, and systemic movement of TVBMV, suggesting that c532 was critical for TVBMV replication. Synonymous substitutions disrupting base pairs C576-G531 and C534-G573 did not affect viral accumulation. After three serial passage inoculation, the accumulation of spontaneous mutant viruses was restored and codons A532G533A534, A532G533G534, a574g575a576, or a574g575g576 of mutants was separately changed to C532G533A534, C532G533G534, C574g575a576, or C574g575g576. Synonymous mutation of R178 and R192 also reduced viral accumulation in N. tabacum plants. Therefore, we concluded that the two consecutive c532-g575 and c574-g533 base pairs played critical roles in TVBMV replication via maintaining the stability of stem-loop structure formed by nucleotides 531 to 576 of CP-coding sequence.