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A single nucleotide change in the overlapping MP and CP reading frames results in differences in symptoms caused by two isolates of Youcai mosaic virus

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Abstract

Two isolates of Youcai mosaic virus (YoMV) were obtained, and their full-length genomic sequences were determined. Full-length infectious cDNA clones of each isolate were generated in which the viral sequence was under the control of dual T7 and 35S promoters for both in vitro transcript production and agro-infiltration. Comparison of the predicted amino acid sequences of the encoded proteins revealed only four differences between the isolates: three in the RNA-dependent RNA polymerase (RdRp) (V383I and M492I in the 125-kDa protein and T1245M in the 182-kDa protein); and one in the overlapping region of the movement protein (MP) and coat protein (CP) genes, affecting only the N-terminal domain of CP (CP M17T). One of the isolates caused severe symptoms in Nicotiana benthamiana plants, while the other caused only mild symptoms. In order to identify the amino acid residues associated with symptom severity, chimeric constructs were generated by combining parts of the two infectious YoMV clones, and the symptoms in infected plants were compared to those induced by the parental isolates. This allowed us to conclude that the M17T substitution in the N-terminal domain of CP was responsible for the difference in symptom severity. The M17T variation was found to be unique among characterized YoMV isolates. A difference in potential post-translational modification resulting from the presence of a predicted casein kinase II phosphorylation site only in the CP of isolate HK2 may be responsible for the symptom differences.

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Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (no. NRF-2017R1A2B4005966).

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Authors and Affiliations

  1. Department of Applied Biology, Chungnam National University, Daejeon, South Korea

    Hye-Kyoung Ju, Ik-Hyun Kim, Wen-Xing Hu, Boram Kim, Go-Woon Choi, Jungkyu Kim & Hyoun-Sub Lim

  2. Department of Horticulture, Chungnam National University, Daejeon, South Korea

    Yong Pyo Lim

  3. Department of Crop Sciences, University of Illinois at Urbana-Champaign, USDA-ARS, Urbana, IL, USA

    Leslie L. Domier

  4. Floral and Nursery Plants Research Unit, United States National Arboretum, USDA-ARS, Beltsville, MD, USA

    John Hammond

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Supplementary material 1 (DOCX 13 kb)

705_2019_4222_MOESM2_ESM.tif

Supplementary material 2 Supplemental Fig. 1 (a) A schematic diagram of the procedure for cloning the YoMV CP into the PVX overexpression vector, using BamHI and MluI. YoMV HK1 and HK2 CPs were inserted separately. (b) Symptoms caused by PVX overexpression constructs (‘empty’ PVX, or PVX expressing HK1 CP = YoMV CP1, or HK2 CP = YoMV CP2) in systemically infected plants of N. tabacum cv. Xanthi and N. benthamiana (TIFF 13380 kb)

705_2019_4222_MOESM3_ESM.tif

Supplementary material 3 Supplemental Fig. 2 Multiple alignment of sequences of selected tobamovirus ORF6 and ORF6 orthologs. (a) Alignment of ORF6 and ORF6 orthologs showing sequence similarities between YoMV isolates and other tobamoviruses. Positions where at least three sequences share the same residue are highlighted in yellow; additional residues conserved between at least three sequences are highlighted in green, with blue, purple and red utilized if additional residue types are conserved in at least two sequences at a particular position within the alignment. The alignment was created using MUSCLE with the default parameters as implemented in Geneious v.11 (https://www.geneious.com, Kearse et al., 2012). (b) Predicted protein-protein binding sites and post-translational modification sites. Note that some sites overlap, and some sequences (KGMMV, ORSV, SFBV, TMGMV, TSAMV, and ZGMMV) have no predicted post-translational modification sites. None of the ORF6 or ORF6 ortholog proteins were predicted to include a nuclear localization signal (NLS). Predictions were generated by submission of the amino acid sequences to the PredictProtein server (https://predictprotein.org/; Rost et al., 2004). Predicted protein-protein binding and post-translational modification sites are indicated as follows: protein-protein binding site, grey highlighting; cyclic-AMP (cAMP)- and cGMP-dependent protein kinase phosphorylation site, red upper-case font; protein kinase C phosphorylation site, underlining; N-myristoylation site, blue font; amidation site, green lower-case font; casein kinase II phosphorylation site, gold font; N-glycosylation site, purple italic font; prenyl group binding site, red lower-case font (TIFF 3131 kb)

Supplementary material 4 (DOCX 13 kb)

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Ju, HK., Kim, IH., Hu, WX. et al. A single nucleotide change in the overlapping MP and CP reading frames results in differences in symptoms caused by two isolates of Youcai mosaic virus. Arch Virol 164, 1553–1565 (2019). https://doi.org/10.1007/s00705-019-04222-z

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