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Hokkaido University Collection of Scholarly and Academic Papers >
Graduate School of Agriculture / Faculty of Agriculture >
Peer-reviewed Journal Articles, etc >
Importin/exportin-mediated nucleocytoplasmic shuttling of cucumber mosaic virus 2b protein is required for 2b's efficient suppression of RNA silencing
| Title: | Importin/exportin-mediated nucleocytoplasmic shuttling of cucumber mosaic virus 2b protein is required for 2b's efficient suppression of RNA silencing |
| Authors: | Kim, Hangil Browse this author | | Shimura, Hanako Browse this author →KAKEN DB | | Sueda, Kae Browse this author | | Masuta, Chikara Browse this author →KAKEN DB |
| Issue Date: | Jan-2022 |
| Publisher: | PLOS |
| Journal Title: | PLoS pathogens |
| Volume: | 18 |
| Issue: | 1 |
| Start Page: | e1010267 |
| Publisher DOI: | 10.1371/journal.ppat.1010267 |
| Abstract: | Author summaryRNA silencing, a small RNA (sRNA)-mediated sequence-specific mechanism, is a primary antiviral response of host plants against viral infection. Argonaute 1 (AGO1), an effector protein of RNA silencing, associates with sRNAs to degrade viral RNAs or silence the genes involved in biotic stresses in cytoplasm; such functions are promoted by its translocation between the nucleus and cytoplasm. To survive host RNA silencing, cucumber mosaic virus (CMV) encodes an RNA silencing suppressor (RSS), 2b protein (2b). 2b plays multiple roles in suppressing RNA silencing by binding to either sRNAs or AGOs, in the nucleus and the cytoplasm. However, several previous reports suggest that 2b exerts its RSS activity mainly when it is in the cytoplasm. Here, we found that the serine residue at position 28 is phosphorylated, and its phosphorylation negatively regulated the RSS activity of 2b by regulating 2b's nuclear/nucleolar localization. Furthermore, we also found that 2bs were dephosphorylated in the nucleus and exported to the cytoplasm in an exportin-dependent manner. Because 2b's transport between the nucleus and cytoplasm is similar to that of AGOs, 2b seems to work in cells as if it is manipulating its nucleocytoplasmic trafficking to track down its targets in the RNA silencing pathway. The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMP alpha]) is predicted to be involved in 2b's nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b's C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b's nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMP alpha-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b's efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm. |
| Type: | article |
| URI: | http://hdl.handle.net/2115/84197 |
| Appears in Collections: | 農学院・農学研究院 (Graduate School of Agriculture / Faculty of Agriculture) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
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