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Cocksfoot mottle sobemovirus coat protein contains two nuclear localization signals

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Abstract

Cocksfoot mottle virus (CfMV) coat protein (CP) localization was studied in plant and mammalian cells. Fusion of the full-length CP with enhanced green fluorescent protein (EGFP) localized to the cell nucleus whereas similar constructs lacking the first 33 N-terminal amino acids of CP localized to the cytoplasm. CP and EGFP fusions containing mutations in the arginine-rich motif of CP localized to the cytoplasm and to the nucleus in plant cells indicating the involvement of the motif in nuclear localization. In mammalian cells, mutations in the arginine-rich region were sufficient to completely abolish nuclear transport. The analysis of deletions of amino acid residues 1–11, 1–22, and 22–33 of CP demonstrated that there were two separate nuclear localization signals (NLS) within the N-terminus—a strong NLS1 in the arginine-rich region (residues 22–33) and a weaker NLS2 within residues 1–22. Analysis of point mutants revealed that the basic amino acid residues in the region of the two NLSs were individually not sufficient to direct CP to the nucleus. Additional microinjection studies with fluorescently labeled RNA and CP purified from CfMV particles demonstrated that the wild-type CP was capable of transporting the RNA to the nucleus. This feature was not sequence-specific in transient assays since both CfMV and GFP mRNA were transported to the cell nucleus by CfMV CP. Together the results suggest that the nucleus may be involved in CfMV infection.

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Acknowledgments

We wish to thank Merike Sõmera for fruitful discussions and Ants Kurg for the initial supply of fluorescent UTP. This work was supported by Estonian Science Foundation grant no. 7363.

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  1. Tiina Tamm

    Present address: Department of General and Microbial Biochemistry, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010, Tartu, Estonia

Authors and Affiliations

  1. Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, 12618, Tallinn, Estonia

    Allan Olspert, Heiti Paves, Raavo Toomela, Tiina Tamm & Erkki Truve

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Correspondence to Erkki Truve.

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Supplementary Fig. 1

The subcellular localization of CfMV CP and Δ1-33CP fused to the C-terminus of EGFP. CP and Δ1-33CP were transiently expressed in onion (upper two rows) and Cos7 (lower two rows) cells. Left panels correspond to EGFP fluorescence (green), middle to nuclear staining with Hoechst (red) and right to overlay. Cells were transfected with constructs coding the proteins indicated on the left, CP corresponds to full length protein. Scale bars represent 50 μm and 10 μm in onion and Cos7 cells, respectively (TIFF 598 kb)

Supplementary Fig. 2

The subcellular localization of CfMV CP mutants R3L and R5X fused to EGFP, transiently expressed in onion epidermal cells. Images from additional independent experiments revealing the consistency of the localization patterns. Upper panels correspond to EGFP fluorescence (green), middle to nuclear staining with EtBr (red) and bottom to overlay. Cells were transfected with constructs coding the proteins indicated on top. Scale bar 50 μm (TIFF 1589 kb)

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Olspert, A., Paves, H., Toomela, R. et al. Cocksfoot mottle sobemovirus coat protein contains two nuclear localization signals. Virus Genes 40, 423–431 (2010). https://doi.org/10.1007/s11262-010-0456-9

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