A Predicted Stem Loop in Coat Protein-Coding Sequence of Tobacco Vein Banding Mosaic Virus Is Required for Efficient...

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 inoculations, the accumulation of spontaneous mutant viruses was restored, and codons A₅₃₂G₅₃₃A₅₃₄, A₅₃₂G₅₃₃G₅₃₄, A₅₇₄G₅₇₅A₅₇₆, or A₅₇₄G₅₇₅G₅₇₆ of mutants were each 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 the stem-loop structures formed by nucleotides 531 to 576 of the CP-coding sequence.