A Predicted Stem Loop in Coat Protein-Coding Sequence 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 C(574)G(575)C(576) coding conserved residue arginine at position 192 (R-192) of tobacco vein banding mosaic virus (TVBMV) CP. Substitution of the codon C(574)G(575)C(576) to A(574)G(575)A(576) or A(574)G(575)G(576), but not C(574)G(575)A(576), C(574)G(575)T(576), or C(574)G(575)G(576), reduced the replication, cell-to-cell movement, and accumulation of TVBMV in Nicotiana benthamiana plants, suggesting that C-574 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 (C-576-G(531), C-532-G(575), C-574-G(533), and C-534-G(573)) were located at the stem. Synonymous substitutions of R-178-codon C(532)G(533)C(534) to A(532)G(533)A(534) and A(532)G(533)G(534), but not C(532)G(533)A(534), C(532)G(533)T(534), or C(532)G(533)G(534), reduced the replication levels, cell-to-cell, and systemic movement of TVBMV, suggesting that C-532 was critical for TVBMV replication. Synonymous substitutions disrupting base pairs C-576-G(531) and C-534-G(573) did not affect viral accumulation. After three serial-passage inoculations, the accumulation of spontaneous mutant viruses was restored, and codons A(532)G(533)A(534), A(532)G(533)G(534), A(574)G(575)A(576), or A(574)G(575)G(576) of mutants were each separately changed to C(532)G(533)A(534), C(532)G(533)G(534), C(574)G(575)A(576), or C(574)G(575)G(576). Synonymous mutation of R-178 and R-192 also reduced viral accumulation in N. tabacum plants. Therefore, we concluded that the two consecutive C-532-G(575) and C-574-G(533) 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.